CN103773771A - Transcription factor system as well as preparation method and application thereof - Google Patents
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Abstract
The invention provides a transcription factor system as well as a preparation method and an application thereof. The invention relates to recombinant lentiviral vectors of three neurogenesis-related transcription factors Ascl1, Brn2 and Pax6 as well as a construction method and an application thereof. In particular, Ascl1, Brn2 and Pax6 target genes are obtained by utilizing a PCR (polymerase chain reaction) method and pGC-LV-GFP is used as a recombinant vector and synthesized DNA fragments of the three target genes are inserted into the recombinant lentiviral vectors, thus constructing GFP (green fluorescent protein) reporter gene built-in recombinant lentivirus plasmids pGC-GFP-Ascl1, pGC-GFP-Brn2 and pGC-GFP-Pax6 respectively containing Ascl1, Brn2 and Pax6. After lentivirus packaging, the three recombinant lentivirus plasmids are used for respectively transfecting a 293T cell and three recombinant lentivirus particles LV-Ascl1, LV-Brn2 and LV-Pax6 are obtained after culture and are concentrated by ultracentrifugation, and the titers of the packaged viruses are determined. Therefore, the transcription factor system can be applied to gene therapy of Alzheimer diseases and provides a new safe and effective therapy path for patients.
Description
Technical field:
The present invention relates to medicine and genetically engineered field.More specifically, the present invention relates to three kinds of neuron regeneration associated transcription factor Ascl1, the recombined lentivirus vector of Brn2 and Pax6 and structure thereof, also relate to three kinds of neuron regeneration associated transcription factor Ascl1, Brn2 and the application of Pax6 recombined lentivirus vector in alzheimer's disease gene therapy.
Background technology:
Alzheimer's disease (AD) claim again senile dementia, is one of whole world nerve degenerative diseases of greatest concern.AD is that the senior cognition dysfunction of a kind of carrying out property and memory function are lost the disease for feature.Research shows, AD sickness rate increases with age growth, and the sickness rate of 65 years old approximately can reach 8% in 0.5%, 85 years old above.Along with China progressively enters aging society, aging trend will make old dementia patients quantity increase, and bring heavy economy and psychological burden to individual, family and society.Although the pathogenesis of AD is not yet completely clear, large quantity research shows amyloid beta deposition, neurofibrillary tangles, and chronic neural inflammation, a large amount of neurocyte degeneration necrosis etc. occur to play a significant role in evolution at AD.Also can effectively stop or reverse alzheimer's disease progression of disease without any a kind of methods for the treatment of at present.Alzheimer's disease was treated still clinically in medicine limitation, stage that curative effect is not good enough.Therefore, developing new treatment technology and method is pendulum problem demanding prompt solution in face of people.
Along with the develop rapidly of molecular biology and bioengineering, make more and more convergence reality of gene therapy central nervous system disease.Alzheimer's disease gene therapy, as a kind of novel treatment means, has become the focus of Neuroscience Research.There are some researches show in recent years, in the somatocyte that the transcription factor of particular combinations is broken up in whole end by certain approach ectopic expression, these somatocyte directly can be induced and are divided into specific functioning cell, complete between cell lineage and change, being the somatocyte that is divided into again function after multipotent stem cells and do not need reprogrammed, is the important breakthrough of gene therapy research.Thereby utilizing external source transcription factor gene treatment alzheimer's disease is a kind of comparatively desirable strategy.
Magdalena research team studies confirm that transcription factor Pax6 crosses in the spongiocyte that is expressed in mouse and can cause that spongiocyte Induction Transformation is neuronic potential (HeinsNetal., 2002).2010, Vierbuchen etc. filter out Ascl1 from 19 specifically expressings in nervous tissue or the gene relevant to cell lineage conversion reprogrammed, Brn2, Myt1l gene, by virus-mediated ectopic expression, is successfully converted into induction type neurocyte by embryo and postpartum mice inoblast, the neurocyte of these inductions can be expressed multiple neuronal specificity albumen, produce action potential, form functional cynapse (Vierbuchenetal., 2010).Heinrich leader's research team finds that by research astroglia cell can directly be divided into Glutamatergic neurone or gamma-hydroxybutyric acid serotonergic neuron proceeding to after transcription factor Neurog2 or Dlx2, confirm for the first time by selecting the neurone (Heinrichetal., 2010) that different transcription factors can be different subtype by direct somatocyte Induction Transformation.Within 2011, Caiazzo research group experimental results show that experiment discovery, human fibroblasts is proceeding to after 3 transcription factor combinations (Ascl1, Nurr1, Lmx1a), directly transdifferentiation is induction type dopaminergic neuron, and there is the function (Caiazzoetal., 2011) that produces Dopamine HCL.2012, Liu etc. test confirmation, people's fibrocyte is expressed to five kinds of transcription factors (Mash1, Ngn2, Sox2 by virus-mediated mistake, Nurr1, Pitx3), can induce it to be converted into dopaminergic neuron, and further rat in vivo test show that it can alleviate the symptom of PD rat model, can be used as the potential therapeutic strategy of disturbances in patients with Parkinson disease (Liuetal., 2012).Karow etc. are crossed transcription factor Sox2 and Mash1 to be expressed in pallium pericyte by retrovirus, it can be programmed and is converted into induction neurone, realize the direct conversion between autogenous cell, further for transcription factor provides possibility (Goritzetal., 2011) for the gene therapy of cental system neurodegeneration.Studies show that, the neuronal function that single transcription factor induction generates may be not perfect, only possesses neurocyte feature, do not possess neuronal function or neuronal function imperfection, therefore can not be used for clinical.And known specific cell is as spongiocyte from the above mentioned, and inoblast etc., need specific transcription factor induction;
Transcription factor Ascl1, Brn2, Pax6 etc. participate in nervous tissue and with cell lineage conversion reprogrammed, and in testing in vitro to other genes involveds, be proved participate in Neural differentiation, regeneration is relevant, but still has great difficulty for gene therapy alzheimer's disease.Its reason may have, first, central nervous system mainly contains neurone and neurogliocyte forms, neurone has non-renewable characteristic, although cell lineage conversion reprogrammed can be converted into body somatic induction the treatment of neurone for central nervous system degeneration, and the neurone that multiple transcription factor combined induction transforms can have that general neurone has feature and function, but select suitable transcription factor to be combined into a difficult problem.At present, although Ascl1, Brn2, the genes such as Pax6 are proved and participate in Neural differentiation in experiment, regeneration is relevant, and there is not yet this three's transcription factor array configuration induction spongiocyte transduction of report is neuronic report; Moreover gene therapy needs suitable, safe genophore, and implement by effective and feasible approach.Slow virus is a kind of retroviral vector, not only can infect the cell of division stage and be incorporated in its genome, but also can infect the cell of non-division stage, and having and hold the advantages such as exogenous goal gene fragment is large, immune response is little, it is widely used as gene therapy vector.Although had remarkable progress but prior art also shows the research of lentiviral vectors, had got long long way to go apart from clinical application.First, the titre of recombinant virus is high not enough, all 10
1tU/ml～10
3between TU/ml, be difficult to reach the needs of application in body; Secondly, due to the biological property of HIV complexity, as conventional Murine retroviral carrier, set up stable HIV vector packaging cell very difficult, the packing cell of having set up is all undesirable.
The present invention utilizes slow virus to set up transcription factor Ascl1 as genophore, Brn2, and Pax6 recombinant slow virus particle, is used for the treatment of alzheimer's disease mouse, observes mouse spatial memory capacity and improves situation.
Summary of the invention:
The object of the invention is to be to provide three kinds of neuron regeneration associated transcription factor Ascl1, Brn2, Pax6, it changes in nervous tissue and to cell lineage the gene that reprogrammed is relevant as specifically expressing, can between inducing cell pedigree, change, there is the ability of induction neuron regeneration.
Another object of the present invention is the preparation method who is to provide three kinds of transcription factor gene recombined lentivirus vectors, by transcription factor Ascl1, Brn2, Pax6 gene is cloned in respectively lentivirus transfer carrier pGC-L-GFP, obtain recombinant slow virus plasmid pGC-L-GFP-Ascl1, pGC-L-GFP-Brn2, pGC-L-GFP-Pax6, and then with packaging plasmid carrier pHelper1.0 and pHelper2.0 cotransfection 293T cell, cultivate and obtain packing recombinant slow virus particle LV-Ascl1, LV-Brn2, LV-Pax6.And through ultracentrifugation concentrating virus particle, measure packaging virus titre.
Another object of the present invention is the bilateral hippocampus that is three kinds of transcription factor recombinant slow virus particles that prepare to inject alzheimer's disease mouse model, observes the change situation of the spatial memory learning capacity of three kinds of rear model mices of vector particles treatment that mix.
To achieve these goals, the present invention adopts following technical measures:
Three kinds of neuron regeneration associated transcription factor Ascl1, Brn2, the preparation method of Pax6, the steps include:
Goal gene Ascl1, Brn2, the DNA fragmentation of Pax6 synthetic: with reference to Genbank mouse Ascl1, Brn2, Pax6 gene order, purpose of design gene primer, extraction, amplification, purifying Ascl1, Brn2, the DNA fragmentation of Pax6.
Goal gene fragment is inserted to the lentivirus transfer carrier pGC-L-GFP with reporter gene GFP, after virus packing, with packaging plasmid carrier pHelper1.0 and pHelper2.0 cotransfection 293T cell, cultivate and obtain packing recombinant slow virus particle LV-Ascl1, LV-Brn2, LV-Pax6.The slow virus LV-GFP that same method obtains in contrast.
By after centrifugal the supernatant liquor of collecting through membrane filtration, further ultracentrifugation obtains the slow virus particle of purifying then, further measures packaging virus titre.
The application aspect alzheimer's disease gene therapy of three kinds of transcription factor recombinant slow virus particles.The steps include:
In order to evaluate three kinds of recombinant slow virus particle LV-Ascl1, LV-Brn2, the effect of LV-Pax6 aspect alzheimer's disease gene therapy, we choose alzheimer's disease mouse model as experimental subjects, observe it through after slow virus granule therapy, the change situation of spatial memory learning capacity.
We are three kinds of recombinant slow virus particle LV-Ascl1, LV-Brn2, and LV-Pax6 mixes (virus titer 1*10 according to the ratio of 1:1:1
8tU/ml), then mixed viral mixed solution is passed through in the mouse bilateral hippocampus of stereotactic injection instrument injection model group, control group gives physiological saline, and model group gives slow virus LV-GFP in contrast.
Latter one week of hybrid virus injection, observe the change situation of each group of mouse spatial memory learning capacity by water maze laboratory, evaluate three kinds of recombinant slow virus particle LV-Ascl1, LV-Brn2, LV-Pax6 is in the effect improving aspect alzheimer's disease spatial memory learning capacity.Fig. 1 be four groups of mouse through recombinant slow virus particle LV-Ascl1, LV-Brn2, water maze laboratory result after LV-Pax6 treatment.
The neurogliocyte of mentioning in the present invention, or be called for short spongiocyte, spongiocyte is more than neurone; at mammals; the ratio of the two is about ten to one, and spongiocyte does not have nerve conduction ability, but neuronic normal activity and substance metabolism are all played an important role.
Beneficial effect:
1, prior art shows, amyloid beta deposition, a large amount of neurogliocyte hyperplasia, neuronal deaths are the main pathological change process in alzheimer's disease generation evolution.The present invention successfully utilizes transcription factor Ascl1 first, Brn2, Pax6 mediation neurogliocyte be converted into induction neurone with and further treat alzheimer's disease.Concrete the present invention packs this three kinds of Ascl1 by slow virus technology, Brn2, and Pax6 gene, and be integrated in the genome of neurogliocyte, bring into play the effect that its Neuronal induction transforms.Can induce neurone transformation tissue culture in body level, improve the space learning memory capability of alzheimer's disease mouse.
What 2, the present invention adopted is gene therapy approach, three kinds of transcription factor Lentivirals that are about to restructuring directly import mouse bilateral hippocampus, we observe mouse space learning memory capability and obviously improve, and untoward reaction is few, effect is affirmed, for treatment of alzheimer provides new thinking.
Although 3, prior art shows that the research of lentiviral vectors has had remarkable progress, has got long long way to go apart from clinical application.First, the titre of recombinant virus is high not enough, all 10
1tU/ml～10
3between TU/ml, be difficult to reach the needs of application in body; Secondly, due to the biological property of HIV complexity, as conventional Murine retroviral carrier, set up stable HIV vector packaging cell very difficult, the packing cell of having set up is all undesirable.And the titre 2*10 of the present invention's restructuring
8tU/ml, has reached the needs that vivo medicine-feeding is applied.
4, lentiviral vectors has the features such as pattern of infection is wide, efficiency is high, immunogenicity is lower, can be for a long time after infection, expression alien gene stably;
Accompanying drawing explanation
Fig. 1. orientation navigation test result analysis figure (escape latency data results) in water maze laboratory;
Fig. 2. the space exploration interpretation figure in water maze laboratory; If Fig. 2 A is for wearing platform number of times data results, Fig. 2 B is fourth quadrant cross-over frequency interpretation of result figure.
Embodiment
Below in conjunction with specific examples, the invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting scope of the present invention.
1: three kind of neuron regeneration associated transcription factor Ascl1 of embodiment, Brn2, the structure of the recombinant slow virus expression vector of Pax6 is according to goal gene Ascl1, Brn2, synthesizing of the DNA fragmentation of Pax6: according to people Ascl1(Genbank accession number: NM_008553), Brn2(Genbank accession number: NM_008899), Pax6(Genbank accession number: NM_008899) and gene order, purpose of design gene primer, its sequence is as follows:
（1）Ascl1：5’-caagagcgcagccttag-3’；3’-gcaaaagtcagtgctgaacg-5’。
（2）Brn2：5’-aataaggcaaaaggaaagcaact-3’；3’-caaaacatcattacctgct-5’；
（3）Pax6：5’-gccagcaacacacctagtca-3’；3’-ggggaaatgagtcctgttga-5’。
Employing contains transcription factor Ascl1, Brn2, the gene fragment of the DNA sequence dna of Pax6 goal gene, this gene fragment is bought and is obtained by human cDNA library (Ying Ji bio tech ltd, Shanghai), to Ascl1, Brn2, Pax6 goal gene carries out pcr amplification, obtains PCR product (PCR test kit is purchased from Takara company).
Pcr amplification reaction system (25ul): 10*buffer2.5ul, primer (10pmol/ul) each 0.5ul, dNTP(10mmol/L) 2ul, archaeal dna polymerase (2.5U/ul) 0.4ul, DNA profiling 1ul, adds distilled water and mends to 25ul; The specification sheets of concrete response procedures reference reagent box.
PCR reaction conditions: 94 ℃ of sex change 5min; (94 ℃ of 45sec, 60 ℃ of 45sec, 72 ℃ of 60sec) 32 circulations; 72 ℃ of 10min.
After reaction finishes, will amplification after be cloned in the lentivirus transfer carrier pGC-L-GFP(that contains reporter gene GFP purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd through the goal gene DNA fragmentation of purifying gained) corresponding site, build recombinant slow virus plasmid pGC-L-GFP-Ascl1, pGC-L-GFP-Brn2, pGC-L-GFP-Pax6(Shanghai JiKai Gene Chemical Technology Co., Ltd builds), for the preparation of slow virus packaging plasmid.
Embodiment 2: recombinant plasmid slow virus packing
1. viral packaging step
The method that Lentiviral is introduced according to (ZhouLetal., 2010) such as Zhou builds.By liposome method Lipofectamine2000 by recombinant slow virus plasmid pGC-L-GFP-Ascl1, pGC-L-GFP-Brn2, pGC-L-GFP-Pax6 and slow virus packaging plasmid pHelper1.0(Invitrogen company, cat.No.11668-500) and pHelper2.0(Invitrogen company, cat.No.11668-500) cotransfection 293T is thin.After 72 hours, collect supernatant, add growth media simultaneously, after 12 hours, again collect supernatant liquor, obtain three kinds of recombinant slow virus particle LV-Ascl1, LV-Brn2, LV-Pax6.
2. results and the concentrated concrete steps of virus
The supernatant liquor that step 1 is obtained is in 4 ℃, and 4000g, after centrifugal 10 minutes, removes cell debris, then through 0.45um membrane filtration supernatant liquor in 40ml ultracentrifugation pipe; Viral crude extract sample is joined in filtering cup, filtering cup is inserted in filtered liquid collection tube, concentrate volume through 4000g ultracentrifugation to virus.After centrifugal end, take out centrifugal device, filtering cup is tipped upside down on sample collection cup, centrifugal force is no more than 1000g, centrifugation time 2 minutes.In sample collection cup, be viral concentrated solution; Viral concentrated solution is shifted out, and packing is preserved in viral pipe ,-80 ℃ of preservations.Get a wherein pipe and do viral biology titer determination.
3. slow virus titre detects concrete steps
Adopt by hole dilution titer assay method, measuring the day before yesterday, for measuring the required cell bed board of titre, every hole adds 4*10
4individual cell, volume is 100ul; According to viral expection titre, prepare 7-10 aseptic Ep pipe, in every pipe, add the serum free medium of 90ul; Get virus stock solution used 10ul to be determined and add in the first pipe, after mixing, get 10ul and be added in second pipe, continue identical operation to last pipe; Choose required cell hole, draw 90ul substratum, abandon, the viral solution that adds 90ul to dilute, puts into incubator and cultivates; After 24 hours, add perfect medium 100u; After 4 days, observe luciferase expression situation.Fluorocyte number reduces along with the increase of extension rate.Titre detected result 2*10
8tU/ml.
The application of 3: three kinds of transcription factor recombinant slow virus particles of embodiment aspect alzheimer's disease gene therapy
Choose 40 adult male C57BL6 mouse, be divided at random four groups, be set to blank group, Alzheimer disease model group, high dosage viral therapy group, low dosage viral therapy group, model group mouse and treatment group give A β 1-42(purchased from--) stereotactic injection is in mouse bilateral hippocampus, blank group gives stroke-physiological saline solution.First three day of A β 1-42 modeling, by three kinds of recombinant slow virus particle LV-Ascl1, LV-Brn2, LV-Pax6 mixes (virus titer 1*10 according to the ratio of 1:1:1
8tU/ml), mixed viral mixed solution 2ul injects treatment group mouse bilateral hippocampus by stereotactic injection instrument, and blank group and model group give respectively the stroke-physiological saline solution of same dosage and slow virus LV-GFP in contrast.
Three kinds of transcription factor recombinant slow virus particle mixed solutions are treated latter one week; four groups of mouse are carried out to the water maze laboratory of seven days by a definite date; record mouse and escape latent period; pass through particular quadrant frequency; pass through the data such as the number of times of platform are set in experiment, carry out statistical study, evaluate this three kinds of recombinant slow virus particle LV-Ascl1; LV-Brn2, LV-Pax6 is in the effect improving aspect alzheimer's disease spatial memory learning capacity.
Result shows: compared with model group mouse, through the mouse of three kinds of transcription factor recombinant slow virus particle mixed solution treatments, escape latency obviously shortens (Fig. 1), wear platform number of times number (Fig. 2 A) and fourth quadrant cross-over frequency (Fig. 2 B) significantly increases, after treatment is described, mouse space learning memory capability significantly strengthens, and curative effect is sure; Meanwhile, experiment mice does not show obvious untoward reaction, and has no dead mouse, has illustrated that this therapeutic modality divides security to be guaranteed.Therefore, the present invention can be applied to the gene therapy of alzheimer's disease, for patient provides new treatment approach safely and effectively.
SEQUENCE LISTING
<110> Nanjing Medical University
<120> transcription factor group and preparation method thereof, application
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213> artificial sequence
<400> 1
caagagcgca gccttag 17
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
<210> 3
<211> 23
<212> DNA
<213> artificial sequence
<400> 3
aataaggcaa aaggaaagca act 23
<210> 4
<211> 19
<212> DNA
<213> artificial sequence
<400> 4
caaaacatca ttacctgct 19
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> 5
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
Claims (6)
1. a transcription factor group, is characterized in that described transcription factor group is made up of Ascl1, Brn2 and Pax6, and its mediation neurogliocyte is converted into induction neurone.
2. a recombined lentivirus vector that contains transcription factor group claimed in claim 1.
3. the construction process of the recombined lentivirus vector of transcription factor group according to claim 2, it is characterized in that, by the plasmid take containing the pGC-L-GFP of green fluorescent protein GFP reporter gene as carrier, insert respectively Ascl1, Brn2, Pax6 gene fragment, build three kinds containing Ascl1, Brn2, the pGC-L-GFP-Ascl1 of Pax6 gene fragment and green fluorescent protein GFP reporter gene, pGC-L-GFP-Brn2, the recombinant plasmid of pGC-L-GFP-Pax6, with packaging plasmid carrier pHelper1.0 and pHelper2.0 cotransfection 293T cell, cultivate and obtain packing recombinant slow virus particle, called after LV-Ascl1, LV-Brn2, LV-Pax6.
4. the construction process of the recombined lentivirus vector of transcription factor group according to claim 3, its feature is realized by following steps:
(1) preparation contains goal gene Ascl1, Brn2, the DNA fragmentation of Pax6;
According to Genbank accession number: NM_008553, Genbank accession number: NM_008899, the gene order that Genbank accession number: NM_008899 records, purpose of design gene primer, its sequence is as follows:
Ascl1：5’-caagagcgcagccttag-3’；3’-gcaaaagtcagtgctgaacg-5’。
Brn2：5’-aataaggcaaaaggaaagcaact-3’；3’-caaaacatcattacctgct-5’；
Pax6：5’-gccagcaacacacctagtca-3’；3’-ggggaaatgagtcctgttga-5’;
(2) amplification, purifying Ascl1, Brn2, the object fragment of Pax6 gene;
(3) prepare recombinant slow virus plasmid pGC-L-GFP-Ascl1, pGC-L-GFP-Brn2, pGC-L-GFP-Pax6 by pGC-L-GFP vector plasmid;
(4), with packaging plasmid carrier pHelper1.0 and pHelper2.0 cotransfection 293T cell, cultivate and obtain packing recombinant slow virus particle, called after LV-Ascl1, LV-Brn2, LV-Pax6.
5. the recombined lentivirus vector of transcription factor group according to claim 2, in its characterization step (4), obtaining slow virus titre is 2*10
8tU/ml.
6. the application of the recombined lentivirus vector of transcription factor group according to claim 2 in preparation treatment Alzheimer medicine.
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| WO2016058537A1 (en) * | 2014-10-17 | 2016-04-21 | 中国科学院上海生命科学研究院 | Uses of ascl1 in inducing astrocyte transdifferentation into functional neurons |
| WO2017214940A1 (en) * | 2016-06-16 | 2017-12-21 | 毛侃琅 | Lentiviral expression vector for specifically promoting high expression of cplx2 gene, and applications thereof |
| CN107828826A (en) * | 2017-12-12 | 2018-03-23 | 南开大学 | A kind of external method for efficiently obtaining NSC |
| CN109512842A (en) * | 2018-12-14 | 2019-03-26 | 南昌大学 | Recombinant slow virus Lenti-Drp1-S579A treats the application in anti-nerve retrograde affection drug in preparation |
| WO2022242756A1 (en) * | 2021-05-21 | 2022-11-24 | The University Of Hong Kong | Compositions and methods of targeting the pax6 signaling pathway to reduce formation of amyloid βeta plaques and neurofibrillary tangles |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016058537A1 (en) * | 2014-10-17 | 2016-04-21 | 中国科学院上海生命科学研究院 | Uses of ascl1 in inducing astrocyte transdifferentation into functional neurons |
| WO2017214940A1 (en) * | 2016-06-16 | 2017-12-21 | 毛侃琅 | Lentiviral expression vector for specifically promoting high expression of cplx2 gene, and applications thereof |
| CN107828826A (en) * | 2017-12-12 | 2018-03-23 | 南开大学 | A kind of external method for efficiently obtaining NSC |
| CN109512842A (en) * | 2018-12-14 | 2019-03-26 | 南昌大学 | Recombinant slow virus Lenti-Drp1-S579A treats the application in anti-nerve retrograde affection drug in preparation |
| WO2022242756A1 (en) * | 2021-05-21 | 2022-11-24 | The University Of Hong Kong | Compositions and methods of targeting the pax6 signaling pathway to reduce formation of amyloid βeta plaques and neurofibrillary tangles |
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Application publication date: 20140507 |