CN108753730A - The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application - Google Patents
The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application Download PDFInfo
- Publication number
- CN108753730A CN108753730A CN201810645564.9A CN201810645564A CN108753730A CN 108753730 A CN108753730 A CN 108753730A CN 201810645564 A CN201810645564 A CN 201810645564A CN 108753730 A CN108753730 A CN 108753730A
- Authority
- CN
- China
- Prior art keywords
- ntf4
- umbilical cord
- stem cells
- mesenchymal stem
- cord mesenchymal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- General Chemical & Material Sciences (AREA)
- Rheumatology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to the umbilical cord mesenchymal stem cells and its construction method of stem cells technology field more particularly to a kind of NTF4 genetic modifications and applications.The present invention provides a kind of umbilical cord mesenchymal stem cells of NTF4 genetic modifications, the umbilical cord mesenchymal stem cells of the NTF4 genetic modifications are overexpressed NTF4 albumen.The umbilical cord mesenchymal stem cells of NTF4 genetic modifications of the present invention had both had the function of that stem cell substituted damaged nerve cell; express NTF4 albumen well again; to reach protection nerve cell; promote the effect of cytothesis and reparation; it is the therapy vector to cerebral arterial thrombosis targeted therapy great potential; the effect that hUC-MSCs can be enhanced, achievees the effect that combination among the strong ones.The experimental results showed that the umbilical cord mesenchymal stem cells of NTF4 genetic modifications are applied in focal cerebral ischemia in rats rat, rat brain damage can be effectively repaired.
Description
Technical field
The invention belongs to the umbilical cord mesenchymal stem cells of stem cells technology field more particularly to a kind of NTF4 genetic modifications and
Its construction method and application.
Background technology
Cerebral arterial thrombosis is a kind of common cardiovascular and cerebrovascular disease, and cerebral arterial thrombosis refers to cerebrovascular disease or whole body
Property disease cause brain blood flow partially or fully to supply obstacle, ischemic, anoxic cause brain tissue necrosis to be liquefied, to function barrier occur
Hinder, shows a major class disease of corresponding neurological symptom and sign.Main Types include that focal cerebral and full brain lack
Blood damages.Clinical treatment cerebral arterial thrombosis mainly uses the measures such as extreme early thrombolysis and neuroprotection both at home and abroad at present, but
Most of patients seriously threatens human health because that could not leave the handicaps such as paralysis, aphasia due to timely thrombolysis more than 3h time windows.
Invention content
In view of this, the present invention provides a kind of umbilical cord mesenchymal stem cells of NTF4 genetic modifications and its construction method and
Using for supplementing the prevention of cardiovascular and cerebrovascular disease.
The specific technical solution of the present invention is as follows：
A kind of umbilical cord mesenchymal stem cells of NTF4 genetic modifications, the umbilical cord mesenchymal stem cells of the NTF4 genetic modifications
It is overexpressed NTF4 albumen.
Preferably, the sequence of the NTF4 genes has such as SEQ ID NO：Sequence shown in 1 or SEQ ID NO：1 replaces one
The nucleotide sequence that a or multiple nucleotide obtain.
The present invention also provides the construction method of the umbilical cord mesenchymal stem cells of NTF4 genetic modifications described in above-mentioned technical proposal,
Include the following steps：
NTF4 gene recombination plasmid carriers are built, then the NTF4 gene recombination plasmids carrier is transfected into pretreated umbilical cord
Mescenchymal stem cell obtains the umbilical cord mesenchymal stem cells of the NTF4 genetic modifications.
Preferably, the NTF4 gene recombination plasmids carrier is the recombined lentivirus vector containing NTF4 genes.
Preferably, the recombined lentivirus vector containing NTF4 genes is by carrying NTF4 genes and CD513B-1 plasmids
Body is connected to be obtained with packaging plasmid packaging recombinant slow virus again.
Preferably, the pretreated umbilical cord mesenchymal stem cells are the umbilical cord of umbilical cord mesenchymal stem cells medium culture
Mescenchymal stem cell；
Glutamine, albumin and gamma-globulin are added in the umbilical cord mesenchymal stem cells culture medium.
Preferably, the recombined lentivirus vector containing NTF4 genes is transfected into the umbilical cord mesenchymal stem cells
Further include：Polybrene is added；
The final concentration of 1 μ μ of g/ml~10 g/ml of the polybrene.
The present invention also provides the umbilical cord mesenchymal stem cells of NTF4 genetic modifications described in above-mentioned technical proposal or above-mentioned technologies
The umbilical cord mesenchymal stem cells of NTF4 genetic modifications made from construction method described in scheme are preparing prevention cardiovascular and cerebrovascular disease medicine
Application in object.
Preferably, the cardiovascular and cerebrovascular disease is cerebral arterial thrombosis.
The present invention also provides a kind of drugs, including the umbilical cord mesenchyma of NTF4 genetic modifications described in above-mentioned technical proposal is dry thin
The umbilical cord mesenchymal stem cells of NTF4 genetic modifications made from construction method described in born of the same parents or above-mentioned technical proposal.
In conclusion the present invention provides a kind of umbilical cord mesenchymal stem cells of NTF4 genetic modifications, the NTF4 genes
The umbilical cord mesenchymal stem cells of modification are overexpressed NTF4 albumen.The umbilical cord mesenchymal stem cells of NTF4 genetic modifications of the present invention both had
There is stem cell to substitute the function of damaged nerve cell, and expression NTF4 albumen well, to reach protection nerve cell, promotes thin
The effect that born of the same parents regenerate and repair, is the therapy vector to cerebral arterial thrombosis targeted therapy great potential, can enhance hUC-
The effect of MSCs, achievees the effect that combination among the strong ones.The experimental results showed that the umbilical cord mesenchymal stem cells of NTF4 genetic modifications are answered
For in focal cerebral ischemia in rats rat, can effectively repair rat brain damage.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is the genophore structure figures of NTF4 in the present invention；
Fig. 2 is that umbilical cord mesenchymal stem cells culture medium and common stem cell media culture umbilical cord mesenchyma are dry in the present invention
The growth curve of cell；
Fig. 3 is that umbilical cord mesenchymal stem cells culture medium and common stem cell media culture umbilical cord mesenchyma are dry in the present invention
The transfection efficiency of cell；
Fig. 4 is the NTF4 albumen extracted in NTF4/hUC-MSCs cell lines and BV/hUC-MSCs cell lines in the present invention
Protein electrophoresis figure；
NTF4/hUC-MSCs cell lines and BV/hUC-MSCs cell lines are extracted after passing on for 5 generations in Fig. 5 present invention
The protein electrophoresis figure of NTF4 albumen；
Fig. 6 is the neurological deficits score after the big rat-tail cell transplantation of focal cerebral ischemia in rats of MCAO in the present invention
As a result；
Fig. 7 is the TCC coloration results after the big rat-tail cell transplantation of focal cerebral ischemia in rats of MCAO for 24 hours in the present invention；
The TCC coloration results that Fig. 8 is 14d after the big rat-tail cell transplantation of focal cerebral ischemia in rats of MCAO in the present invention.
Specific implementation mode
The present invention provides a kind of umbilical cord mesenchymal stem cells of NTF4 genetic modifications and its construction method and applications, are used for
The prevention of cardiovascular and cerebrovascular disease is supplemented.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Mode is only some embodiments of the invention, rather than whole embodiments.Based on the embodiment in the present invention, originally
The every other embodiment that field those of ordinary skill is obtained without making creative work, belongs to this hair
The range of bright protection.
A kind of umbilical cord mesenchymal stem cells of NTF4 genetic modifications, the umbilical cord mesenchymal stem cells of NTF4 genetic modifications cross table
Up to NTF4 albumen.
Neurotrophic factor 4 (neurotrophin 4, NTF4) is a kind of important neurotrophic factor, NTF4 and nerve
Growth factor (Nerve growth factor, NGF), brain-derived neurotrophic factor (brain derived
Neurotrophic factor, BDNF) there is similar gene order structure and biological activity, in mammalian nervous system
It plays an important role during development, differentiation and injury repair of system etc., growth, the differentiation of neuron can be promoted, promote nerve
The physiological actions such as injury repair and regeneration, have potential application in terms for the treatment of a variety of neurological disorders.Research is found
NTF4 is stronger to the facilitation ratio BDNF of aixs cylinder ganglion growth, and NTF4 can prevent intracellular membranes after neure damage
Flowing adjusts calcium ion level to save neuronal degeneration necrosis, while can pass through the expression of change cell adhesion molecule
Promote the regeneration of injury region nerve, there is the performance for resisting ischemia injury.
Umbilical cord mesenchymal stem cells are the important members of stem cell line, derive from neonatal umbilical cord tissue, belong to more
It can stem cell.
In the present invention, umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells (human umbilical cord
Mesenchymal stem cells, hUC-MSCs).HUC-MSCs convenient material drawings, derive from a wealth of sources, and application is not striven by ethics
By and limitation, while draw materials to donor hurtless measure, do not influenced by donor age factor, Isolation and culture is simple, amplification it is fast
Speed, immunogenicity is relatively low, and no oncogenicity can also be used as the cell carrier of gene therapy.Item is specifically induced in vivo or in vitro
Under part, hUC-MSCs can be divided into Various Tissues cell, still have multi-lineage potential after continuous passage culture and freezen protective,
It is the ideal seed cell of cellular transplantation therapy, is widely used to the treatment of clinical a variety of diseases at present.
HUC-MSCs can express the relevant neurotrophic factor of a variety of rush nerve growths, as BDNF, epidermal growth factor,
Glial cell line-derived neurotrophic factor, hepatocyte growth factor, NT-3 neurotrophic factors, vascular endothelial growth factor, class
Insulin No.1 growth factor, NTF4, nerve growth factor etc., play therapeutic effect in central nervous system disease.It is moving
After implanting, on the one hand the sustainable expression of these factors improves microenvironment in local cerebral, repairs the lesion of damage, another party
Face starts the activation and differentiation of Endogenous neural stem cells, promotes the survival and regeneration of neuron, makees to play CO2 laser weld
With.The umbilical cord mesenchymal stem cells of NTF4 genetic modifications of the present invention are overexpressed NTF4 albumen, are filled between the umbilical cord of NTF4 genetic modifications
Matter stem cell had both had the function of that stem cell substituted damaged nerve cell, and the umbilical cord mesenchymal stem cells of NTF4 genetic modifications can
It is differentiated to form new neuron in vivo, newborn neuron moves to affected area and replaces dead neuronal, also, NTF4 immediately
The umbilical cord mesenchymal stem cells of genetic modification can be good at expressing NTF4 albumen, to reach protection nerve cell, promote cell
Regeneration and the effect repaired, are the therapy vectors to cerebral arterial thrombosis targeted therapy great potential, can enhance hUC-MSCs
Effect, achieve the effect that combination among the strong ones.Subsequent experimental the result shows that, the umbilical cord mesenchymal stem cells of NTF4 genetic modifications are answered
For in focal cerebral ischemia in rats rat, can effectively repair rat brain damage.
In the present invention, the umbilical cord mesenchymal stem cells of NTF4 genetic modifications, which are stablized, is persistently overexpressed NTF4 albumen.
In the present invention, the sequence of NTF4 genes has such as SEQ ID NO：Sequence shown in 1 or SEQ ID NO：1 replaces one
Or the nucleotide sequence that multiple nucleotide obtain.
The present invention also provides the construction methods of the umbilical cord mesenchymal stem cells of above-mentioned technical proposal NTF4 genetic modifications, including
Following steps：
NTF4 gene recombination plasmid carriers are built, then NTF4 gene recombination plasmid carriers are transfected between pretreated umbilical cord
Mesenchymal stem cells obtain the umbilical cord mesenchymal stem cells of NTF4 genetic modifications.
In the present invention, NTF4 gene recombination plasmid carriers are the recombined lentivirus vector containing NTF4 genes.
In the present invention, the recombined lentivirus vector containing NTF4 genes is by by NTF4 genes and CD513B-1 plasmid vectors
It is connected and is obtained again with packaging plasmid packaging recombinant slow virus.
In the present invention, umbilical cord mesenchymal stem cells are preferably P2 for umbilical cord mesenchymal stem cells.
In the present invention, the construction method of the umbilical cord mesenchymal stem cells of NTF4 genetic modifications specifically includes：
A) NTF4 genes are obtained by PCR, double digestion is carried out to CD513B-1 plasmid vectors with EcoR I and BamH I, then
CD513B-1 plasmid vectors after digestion are connect with NTF4 genes with T4DNA ligases, obtain recombinant C D513B-NTF4 matter
Grain；
B) recombinant C D513B-NTF4 plasmids and packaging plasmid pMD2G and pCMVdR8 are subjected to slow virus incasing cells
The transfection of HEK293FT obtains the recombined lentivirus vector containing NTF4 genes；
C) recombined lentivirus vector containing NTF4 genes is transfected to P2 for umbilical cord mesenchymal stem cells, obtains NTF4 bases
Because of the umbilical cord mesenchymal stem cells of modification.
In the present invention, pretreated umbilical cord mesenchymal stem cells are between the umbilical cord of umbilical cord mesenchymal stem cells medium culture
Mesenchymal stem cells；
Glutamine, albumin and gamma-globulin are added in umbilical cord mesenchymal stem cells culture medium.
Umbilical cord mesenchymal stem cells culture medium is prepared by adding glutamine, albumin and gamma-globulin in the medium
It obtains.
The stem cell media being commonly used is usually the first generation and second generation culture medium, is formulated indefinite, Chang Han
There are an animal derived components, difference is larger between product batches, cannot effectively maintain the dryness of stem cells, stem cell can passage number
It is low.Umbilical cord mesenchymal stem cells culture medium of the present invention is without adding serum or serum substitute, culture medium specific chemical components, nothing
Serum, non-animal derived ingredient are more suitable for clinical research use, ensure that umbilical cord mesenchyma is dry thin in cell culture, succeeding generations
Born of the same parents can keep its potential to Multidirectional Differentiation.
In addition, currently employed slow virus packing technique, needs to carry out the medium supernatant containing virus liquid dense
It is used after contracting, either content or PCR methods measure disease to the activity of associated viral protein in generally use measurement virion
Malicious titre, however preceding kind of method measures the titre come and be inaccurate and the latter needs design primer, increase operating procedure and when
Between, while different cell there are certain requirements the titre of virus liquid, excessive concentration or too low can be generated to the transfection of cell
It influences, the sense that stem cell is more advantageous to slow virus is recombinated between people's umbilical cord of umbilical cord mesenchymal stem cells medium culture of the present invention
Dye, reduces supernatant (virus liquid) concentration step, slow-virus transfection can be carried out immediately after collecting supernatant, improves virus and turns
Contaminate efficiency.
The concentration of a concentration of 200mg/L~500mg/L of glutamine, glutamine are preferably 300mg/L.
A concentration of 20mg/mL of albumin.
The concentration of a concentration of 0.05mg/mL~0.25mg/mL of gamma-globulin, gamma-globulin are preferably 0.1mg/mL.
In the present invention, the recombined lentivirus vector containing NTF4 genes is transfected into umbilical cord mesenchymal stem cells and is also wrapped
It includes：Polybrene is added；
The final concentration of the final concentration of 1 μ g/ml~10 μ g/ml of polybrene, polybrene are preferably 5 μ g/ml.
Polybrene by neutralizing the electrostatic repulsion between cell surface sialic acid and virion to promote suction-operated,
The transfection efficiency of slow virus can be improved, but it to the toxic effect of cell, different cells are different to its susceptibility, therefore different
The best use concentration of cell polybrene and time are all different.The present invention uses the polybrene of final concentration of 5 μ g/ml,
Slow-virus transfection can be effectively facilitated, while the influence of recombination stem cell is smaller between people's umbilical cord.
The present invention also provides the umbilical cord mesenchymal stem cells or above-mentioned technical proposal of above-mentioned technical proposal NTF4 genetic modifications
The umbilical cord mesenchymal stem cells of NTF4 genetic modifications made from construction method are preparing answering in preventing cardiovascular and cerebrovascular diseases medicament
With.
In the present invention, cardiovascular and cerebrovascular disease is cerebral arterial thrombosis.
The present invention also provides a kind of drug, including the umbilical cord mesenchymal stem cells of above-mentioned technical proposal NTF4 genetic modifications or
The umbilical cord mesenchymal stem cells of NTF4 genetic modifications made from above-mentioned technical proposal construction method.
For a further understanding of the present invention, with reference to specific embodiment, the present invention will be described in detail.
The structure of embodiment 1NTF4 gene recombination plasmid carriers
(1) PCR obtains NTF4 genes
Using pcDNA3.0-NTF4-Flag plasmids as template, with F:5'CGGAATTCCGATGGAGGGAGAGG 3',R:5'
CGGGATCCCGTCAGGCCCGGCC 3 ' is that primer amplification NTF4 encodes complete sequence.PCR response parameters are：94 DEG C of pre-degenerations
2min；98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 recycle；72 DEG C of extension 1min, 10 DEG C of heat preservations.With
The identification of 1% agarose gel electrophoresis and separation product, recycle using DNA gel QIAquick Gel Extraction Kit and purify NTF4 amplified fragments,
Target fragment size about 633bp.Wherein, pcDNA3.0-NTF4-Flag plasmids are preserved by laboratory, agarose and DNA
Marker is purchased from Beijing Quan Shijin biotech firms, and the used archaeal dna polymerase of PCR amplification uses KOD exo+ polymerases, purchased from east
Paj Shanghai bio tech ltd, DNA gel QIAquick Gel Extraction Kit are purchased from Sai Mofei companies, and primer is synthesized by Suzhou gold only intelligence
Company completes.
(2) preparation of recombinant C D513B-NTF4 plasmids
Double digestion, DNA gel electrophoresis recovery purifying, by enzyme are carried out to CD513B-1 plasmid vectors with EcoRI and BamH I
The amplified fragments of CD513B-1 plasmid vectors and NTF4 after cutting are connected with T4DNA ligases, and NTF4 is inserted into CD513B-1 matter
The recombinant C D513B-NTF4 plasmids of structure are transferred to bacillus coli DH 5 alpha impression by I site of I sites EcoR and BamH of grain carrier
State cell, then Escherichia coli are coated on the tablet containing ampicillin (AmpR), it is inverted 37 DEG C of incubators and stays overnight.It carries out
After bacterium solution PCR and double digestion identification are correct, sequencing identification is carried out.The result shows that choosing colony is the positive.Fig. 1 is in the present invention
The coding complete sequence of NTF4, bacterium solution PCR and sequencing result show the sequence of the nucleotide of recombinant C D513B-NTF4 plasmids with
The sequence of people NTF4 is identical in GeneBank.Using the plasmid purification kit extraction of Sai Mofei companies and plasmid purification, obtain
High-quality recombinant C D513B-NTF4 plasmids.CD513B-1 plasmid vectors are preserved by laboratory, bacillus coli DH 5 alpha competent cell
Purchased from Beijing Quan Shijin biotech firms, restriction enzyme EcoR I and BamH I are purchased from U.S. New England Biolabs
Company, sequencing are completed by Suzhou Jin Weizhi companies.
(3) packaging of slow virus
Slow virus incasing cells HEK293FT condition of culture is the H-DMEM for adding 10% fetal calf serum of volume fraction, transfection
The previous day passes on bed board using 6cm culture dishes, is transfected within second day, and HEK293FT cells reach 70% fusion when transfection, point
2.5 μ g CD513B vector and 2.5 μ g recombinant C D513B-NTF4 plasmids and packaging plasmid 1.25 μ g pMD2G and 1.25 are not taken
μ g pCMVdR8 are mixed in without serum and dual anti-opti-MEM with Lipofectamine 2000, incubation at room temperature
15min after forming 2000 compounds of DNA Lipofectamine, transfects HEK293FT cells, is changed to containing serum after 6h
Normal incubation medium changes liquid afterwards for 24 hours, and culture medium is collected after 48h, and 3000rpm centrifuges 15min, and Aspirate supernatant is obtained containing NTF4
The recombined lentivirus vector CD513B-NTF4 recombined lentivirus vectors and blank slow virus carrier CD513B vector of gene, add
Enter into isometric fetal calf serum, mixing, -80 DEG C of preservations after packing.Wherein, H-DMEM culture mediums and fetal calf serum are purchased from U.S.
Gibco companies of state, Lipofectamine 2000 are purchased from Invitrogen companies,
The preparation of 2 umbilical cord mesenchymal stem cells of embodiment
A moon umbilical cord for normal pregnancy Cesarean esction healthy fetus is taken fully, 2 are washed with 0.9% sodium chloride injection of mass fraction~
3 times, then 2min will be impregnated in 75% alcohol of volume fraction of umbilical cord after filtration, then by 0.9% chloride injection of umbilical cord
Liquid washs 2~3 times, and washing is abundant.Umbilical cord is finally cut into the small hop count sections of about 1cm~3cm, navel artery and vein is rejected with pincers,
And magnificent Tong Shi glue is torn, cut into 1mm3~4mm3Tissue be homogenized block.Centrifuge washing tissue is homogenized block, according to magnificent Tong Shi glue
Quality determines culture bottle number, and per T75 bottles, inoculation 0.5g~0.6g tissues homogenate block, adds 8ml~10ml prepared navels
Interband mesenchymal stem cell media sets 37 DEG C, 5%CO2Saturated humidity incubator in cultivate.It is spaced 5 days~6 days during culture
It changes the liquid once, when cell fusion degree reaches 70%~80%, pancreatin digestion, and press 1：5 are passed on.It is continuous to cultivate to P2 generations
Afterwards, cell surface marker is detected using Flow cytometry, the results showed that comply fully with human umbilical cord mesenchymal stem cells spy
Property, it is placed in liquid nitrogen and freezes.Wherein, umbilical cord mesenchymal stem cells culture medium by adding 300mg/L glutamy in the medium
Amine, 20mg/mL albumin and 0.1mg/mL gamma-globulins are prepared to obtain.The present embodiment culture medium is purchased from Amada Co., Ltd.'s biology
Science Institute.
In the present embodiment, the culture of umbilical cord mesenchymal stem cells is also carried out using common stem cell media.Please refer to figure
2, for the life of umbilical cord mesenchymal stem cells culture medium and common stem cell media culture umbilical cord mesenchymal stem cells in the present invention
Long curve.Fig. 2 shows compared with common stem cell media, the navel through umbilical cord mesenchymal stem cells medium culture of the present invention
Band mescenchymal stem cell growth rate is considerably higher.
The structure of the umbilical cord mesenchymal stem cells of embodiment 3NTF4 genetic modifications
Take P2 for human umbilical cord mesenchymal stem cells from liquid nitrogen, recovery passes on bed board using 6cm culture dishes, waits for people's umbilical cord
When mescenchymal stem cell is grown to 70% cell fusion, the culture medium in culture dish is sucked, it is sick slowly to be separately added into CD513B vector
Venom and CD513B-NTF4 slow virus venom 1mL and base is trained in equal volume, while polybrene is added, final concentration of the 5 of polybrene
μ g/ml set 37 DEG C, volume fraction 5%CO2Cell incubator in overnight incubation.It discards infection liquid afterwards for 24 hours and exchanges for fresh complete
Full culture medium continues to cultivate, and fluorescence microscope detects slow-virus transfection efficiency, observes hU-MSCs in fluorescence microscope after 48h
Under send out green fluorescence, show to infect successfully, then passed on, add puromycin, the final concentration of 2 μ g/ml of puromycin~
10 μ g/ml screen the navel of umbilical cord mesenchymal stem cells NTF4/hUC-MSCs and blank modification that 1 week obtains NTF4 genetic modifications
Stable cell lines with mescenchymal stem cell BV/hUC-MSCs.
In the present embodiment, also slow virus is carried out using the umbilical cord mesenchymal stem cells of common stem cell media culture and turned
Dye.Referring to Fig. 3, for umbilical cord mesenchymal stem cells culture medium in the present invention and common stem cell media culture umbilical cord mesenchyma
The transfection efficiency of stem cell.In Fig. 3, compared with common stem cell media group, * p < 0.05, significant difference.Fig. 3 tables
Bright, compared with common stem cell media, the umbilical cord mesenchyma through umbilical cord mesenchymal stem cells medium culture of the present invention is dry thin
Dysuria with lower abdominal colic contaminates the transfection efficiency higher of slow virus.
Embodiment april protein trace detection method detects tables of the NTF4 in the umbilical cord mesenchymal stem cells of NTF4 genetic modifications
It reaches
The BV/hUC-MSCs cells of the NTF4/hUC-MSCs cell lines and blank modification of expression NTF4 are stablized in extraction respectively
Total protein in system is used in combination BCA methods to measure the albumen concentration of institute's leach protein, and albumen concentration loading is consistent, SDS-PAGE polypropylene
Acrylamide gel electrophoresis is gone to the albumen on gel on pvdf membrane by electricity after electrophoresis, 5% skimmed milk power room of mass fraction
Warm shaking table closes 2h, is incubated rabbit-anti people NTF4 antibody (1 respectively：1000 dilutions) and internal reference rabbit-anti people β-tubulin antibody (1：
5000 dilutions), 4 DEG C of overnight incubations, PBST shaking tables wash pvdf membrane (3 times, each 5min), the goat of incubation at room temperature HRP labels
Anti-rabbit antibody (1：2000 dilution) 2h, PBST shaking tables wash pvdf membrane (3 times, each 5min), use ECL luminescent solutions soak PVDF
Result is observed in film, exposure.Referring to Fig. 4, for NTF4/hUC-MSCs cell lines and BV/hUC-MSCs cells in the present invention
The protein electrophoresis figure of the NTF4 albumen extracted in system, the results showed that the NTF4 protein expressions of NTF4/hUC-MSCs cell lines are apparent
Higher than the NTF4 protein expressions of BV/hUC-MSCs cell lines.Referring to Fig. 5, for the present invention in NTF4/hUC-MSCs cell lines and
BV/hUC-MSCs cell lines pass on the protein electrophoresis figure of the NTF4 albumen extracted after 5 generations, the results showed that, NTF4 genetic modifications
Umbilical cord mesenchymal stem cells, which are stablized, is persistently overexpressed NTF4 albumen.Wherein, protein immunoblot lysate, determination of protein concentration examination
Agent box is purchased from green skies biotechnology research institute, and ECL luminescent solutions are purchased from Millipore companies of the U.S., rabbit-anti people NTF4 antibody, rabbit
The goat anti-rabbit antibodies of anti-human β-tubulin antibody and HRP labels are purchased from Abcom companies of the U.S., and skimmed milk power is purchased from Wanda Mountain
Company, loading buffer are purchased from Beijing Quan Shijin biotech firms, and general chemical reagent is that domestic analysis is pure.
The foundation of 5 cerebral arterial thrombosis animal model of embodiment
It, will be big using Zea Longa ' the s line brush of modified form by SD rats after intraperitoneal injection yellow Jackets anesthesia
Mouse four limbs are fixed on operating table, neck preserved skin, disinfection, a notch for being about 2cm are done in neck center, with tweezers and scissors point
Separate out right carotid (common carotid crtery, CCA), external carotid artery (external carotid artery,
) and internal carotid (InternalCarotid Artery, ICA) ECA.CCA, ECA are ligatured with the silk thread of diameter 4.0mm, in ICA
A untwisting is made a call at place；A " V " shape notch is cut in the nearly crotches of ICA, and (loop length is according to rat body weight by Insert Here choke line
Depending on), it directes reach and sends out at arteria cerebri media, ligature ICA, the end of skin suture, choke line is exposed in vitro.It blocks on the right side of rat
Arteria cerebri media establishes the focal brain of middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO)
Ischemia model.Obstruction line front end is fallen back on into neck after ischemic 2h, and cuts off and exposes external choke line, carries out ischemia-reperfusion.
There is cervical sympathetic paralysis syndrome (Horner signs) in clinical follow animal, carries and receives buckling in tail left fore, when walking to the left
Circle is drawn in side, falls down to the left when serious, and left fore grip weakens, and carries out improvement neurological deficits score standard
(Modifiedneurological severity score, mNSS) scores (≤9 point), brain is taken for 24 hours after model foundation, is cut into
The coronal section of about 1mm thickness is tested with triphenyltetrazolium chloride (triphenyltetrazolium chloride, TTC) dyeing
The validity for demonstrate,proving modeling method is turn-taked when being walked with rat to the right as modeling success.Wherein, SD rats are that healthy adult SD is male
Property rat, quality be 220g~250g, totally 40, be purchased from Nantong University's Experimental Animal Center；Choke line is purchased from Beijing Sha Dongsheng
Object Technology Co., Ltd..
6 animal packet of embodiment and cell transplantation
The 2nd day after modeling success, the focal cerebral ischemia in rats rat 24 for being successfully established MCAO is only randomly divided into NTF4/
HUC-MSCs groups, BV/hUC-MSCs groups and Control groups, every group 8.It is digested with the pancreatin room temperature of mass fraction 0.25%
NTF4/hUC-MSCs and BV/hUC-MSCs, fully piping and druming are mixed into single cell suspension, and cell density is adjusted to 1 with physiological saline
×105A/μ L.The tail vein of exposure focal cerebral ischemia in rats rat will extract 100 μ L NTF4/hUC-MSCs cell suspensions
It is inserted into NTF4/hUC-MSCs groups and BV/hUC-MSCs group rats respectively with the syringe of 100 μ L BV/hUC-MSCs cell suspensions
Tail vein in, slowly inject, carry out cell transplantation.Control groups inject 100 μ L physiological saline, injection site and operation side
Method is the same as NTF4/hUC-MSCs groups and BV/hUC-MSCs groups.After injecting, injection site, while intramuscular injection penicillin 100,000 are sterilized
U prevents infection, sub-cage rearing.
7 neurological deficits score of embodiment and result
With reference to mNSS standards of grading, 6h, for 24 hours, score after 3d, 7d, 14d is transplanted respectively at rat cell.Scoring mark
Standard includes movement, feels, the test of four aspects of balance and reflection, and total score 18 divides.13-18 points are severe injury；7-12 points
For moderate lesion；2-6 points are minor injury.Specific standards of grading are as follows：
One, movement checks (6 points)
1. tail tests (3 points).
0 point：Limbs not buckling
1 point：Forelimb is bowed (Ipsilateral) in the wrong
1 point：Hind leg is bowed (Ipsilateral) in the wrong
1 point：Head is deviated from tailing axle in 30s>10°
2. amplifying mouse in (normal 0 point, 3 points maximum) on floor
0 point：Normal walking
1 point：It is unable to straight line moving
2 points：It turn-takes paralysis side
3 points：It falls down paralysis side
Two, feel to check (2 points)
1 point：Vision, tactile inspection (including antenna and blunt stick touch reaction)
1 point：Deep sensory inspection (by suffering limb to table side along pushing away, suffering limb contraction of muscle is with far from edge)
Three, balance beam inspection (normal 0 point, 6 points maximum) (crossbeam long 1m, wide 10cm, liftoff 1m high)
0 point：Stable posture balances
1 point：One side of crossbeam can be caught
2 points：Crossbeam can be embraced, but crossbeam can be left there are one limbs
3 points：It can embrace crossbeam, but there are two limbs leave crossbeam or the rotation and more than 60s on crossbeam
4 points：It attempts to keep balance on crossbeam, though it can finally be fallen more than 40S
5 points：It attempts to keep balance on crossbeam, though it can finally be fallen more than 20s
6 points：It can not balance or be suspended on crossbeam and be fallen in 20s
Four, defect and abnormal movement (4 points) are reflected
1 point：Auricle reflex (head tremor when contacting duct)
1 point：Corneal reflection (eyelid is closed when touching cornea with cotton thread)
1 point：Shock reflection (burst noise scaring sees whether to dodge)
1 point：Epileptic attack, muscle spasmus, myodystony
Respectively at rat cell transplanting after 6h, for 24 hours, 3d, 7d and 14d carried out neurological deficits score, please refer to figure
6, for the neurological deficits score result after the big rat-tail cell transplantation of focal cerebral ischemia in rats of MCAO in the present invention.Fig. 6
In, compared with Control groups, * p < 0.05, significant difference, * * p < 0.01 have pole significant difference；With BV/hUC-
MSCs groups are compared, #p < 0.05, significant difference.The result shows that BV/hUC-MSCs groups when 3d, 7d and 14d after cell transplantation
Control groups are substantially less than with the neurological deficits score of NTF4/hUC-MSCs groups, NTF4/ when 7d and 14d after cell transplantation
The neurological deficits score of hUC-MSCs groups is substantially less than BV/hUC-MSCs groups.
Embodiment 8TTC dyeing and result
Every group takes rat 6, excessive anesthesia to put to death.Overhead skin and cranium are peelled off, brain is exposed.It is light with tweezers
Brain is beaten easily out, its surface hematocele of normal saline flushing, filter paper blots excessive moisture, brain is then put into -80 DEG C of refrigerators, cold
It is taken out after freezing 5min, does continuous coronal section, totally 5~6, every 1mm thickness.Brain piece is placed in mass fraction 2%2,3,5- tri-
In tetraphenylphosphonium chloride tetrazole (TTC) solution, 30min is incubated in 37 DEG C of insulating boxs.It takes pictures to brain piece after dyeing, measures cerebral infarction dead volume
Product.Wherein, 2,3, 5-Triphenyltertrazoliumchloride (TTC) is purchased from Sigma Co., USA.
Brain tissue TTC dyeing normal structures are red, and white is presented in injury tissue.Referring to Fig. 7, for MCAO in the present invention
The big rat-tail cell transplantation of focal cerebral ischemia in rats after TCC coloration results for 24 hours.Referring to Fig. 8, for MCAO in the present invention
The TCC coloration results of 14d after the big rat-tail cell transplantation of focal cerebral ischemia in rats.BV/hUC-MSCs groups and NTF4/hUC-MSCs
Group 24d and 14d after cell transplantation carry out the detection of cerebral infarction volume, when after cell transplantation for 24 hours, BV/hUC-MSCs groups with
The cerebral infarction volume of NTF4/hUC-MSCs groups is substantially less than Control groups, and the cerebral infarction volume of NTF4/hUC-MSCs groups is notable
Less than BV/hUC-MSCs groups；After cell transplantation when 14d, Control groups and BV/hUC-MSCs group brain tissue ischemias position occur
Atrophy reduces, and NTF4/hUC-MSCs group brain tissue structures are normal.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Nantong Lu hundred million gives birth to bio tech ltd
<120>The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application
<141> 2018-06-20
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 633
<212> DNA
<213> Homo sapiens
<400> 1
atgctccctc tcccctcatg ctccctcccc atcctcctcc ttttcctcct ccccagtgtg 60
ccaattgagt cccaaccccc accctcaaca ttgccccctt ttctggcccc tgagtgggac 120
cttctctccc cccgagtagt cctgtctagg ggtgcccctg ctgggccccc tctgctcttc 180
ctgctggagg ctggggcctt tcgggagtca gcaggtgccc cggccaaccg cagccggcgt 240
ggggtgagcg aaactgcacc agcgagtcgt cggggtgagc tggctgtgtg cgatgcagtc 300
agtggctggg tgacagaccg ccggaccgct gtggacttgc gtgggcgcga ggtggaggtg 360
ttgggcgagg tgcctgcagc tggcggcagt cccctccgcc agtacttctt tgaaacccgc 420
tgcaaggctg ataacgctga ggaaggtggc ccgggggcag gtggaggggg ctgccgggga 480
gtggacagga ggcactgggt atctgagtgc aaggccaagc agtcctatgt gcgggcattg 540
accgctgatg cccagggccg tgtgggctgg cgatggattc gaattgacac tgcctgcgtc 600
tgcacactcc tcagccggac tggccgggcc tga 633
Claims (10)
1. a kind of umbilical cord mesenchymal stem cells of NTF4 genetic modifications, which is characterized in that between the umbilical cord of the NTF4 genetic modifications
Mesenchymal stem cells are overexpressed NTF4 albumen.
2. umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that the sequence of the NTF4 genes has such as
SEQ ID NO：Sequence shown in 1 or SEQ ID NO：1 replaces the nucleotide sequence that one or more nucleotide obtain.
3. the construction method of the umbilical cord mesenchymal stem cells of NTF4 genetic modifications described in claims 1 or 2, which is characterized in that packet
Include following steps：
NTF4 gene recombination plasmid carriers are built, then will be filled between the pretreated umbilical cord of NTF4 gene recombination plasmids carrier transfection
Matter stem cell obtains the umbilical cord mesenchymal stem cells of the NTF4 genetic modifications.
4. construction method according to claim 3, which is characterized in that the NTF4 gene recombination plasmids carrier be containing
The recombined lentivirus vector of NTF4 genes.
5. construction method according to claim 4, which is characterized in that the recombined lentivirus vector containing NTF4 genes
It is obtained again with packaging plasmid packaging recombinant slow virus by the way that NTF4 genes are connected with CD513B-1 plasmid vectors.
6. construction method according to claim 3, which is characterized in that the pretreated umbilical cord mesenchymal stem cells are navel
The umbilical cord mesenchymal stem cells of interband mesenchymal stem cell media culture；
Glutamine, albumin and gamma-globulin are added in the umbilical cord mesenchymal stem cells culture medium.
7. construction method according to claim 5, which is characterized in that carry the recombinant slow virus containing NTF4 genes
Body is transfected further includes into the umbilical cord mesenchymal stem cells：Polybrene is added；
The final concentration of 1 μ μ of g/ml~10 g/ml of the polybrene.
8. the umbilical cord mesenchymal stem cells of NTF4 genetic modifications described in claims 1 or 2 or claim 3 to 7 any one institute
The umbilical cord mesenchymal stem cells of NTF4 genetic modifications made from construction method are stated in preparing prevention cardiovascular and cerebrovascular diseases medicament
Using.
9. application according to claim 8, which is characterized in that the cardiovascular and cerebrovascular disease is cerebral arterial thrombosis.
10. a kind of drug, which is characterized in that include the umbilical cord mesenchymal stem cells of NTF4 genetic modifications described in claims 1 or 2
Or the umbilical cord mesenchymal stem cells of NTF4 genetic modifications made from construction method described in claim 3 to 7 any one.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810645564.9A CN108753730A (en) | 2018-06-21 | 2018-06-21 | The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810645564.9A CN108753730A (en) | 2018-06-21 | 2018-06-21 | The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108753730A true CN108753730A (en) | 2018-11-06 |
Family
ID=63979983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810645564.9A Pending CN108753730A (en) | 2018-06-21 | 2018-06-21 | The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108753730A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609554A (en) * | 2018-11-29 | 2019-04-12 | 南方医科大学 | A kind of umbilical cord mesenchymal stem cells and its preparation method and application of mir338 gene silencing |
| CN111235115A (en) * | 2020-02-25 | 2020-06-05 | 南京鼓楼医院 | Recombinant mesenchymal stem cell, preparation method thereof and application thereof in preparing medicine for treating acute liver failure |
| CN113430231A (en) * | 2021-04-20 | 2021-09-24 | 华夏源细胞工程集团股份有限公司 | Method for efficiently infecting human umbilical cord mesenchymal stem cells by using pseudolentivirus vector |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333542A (en) * | 2007-06-25 | 2008-12-31 | 何星儒 | Process for transplanting and repairing retina damnification in eye by gene modified mesenchyme stem cell |
| CN101857854A (en) * | 2009-04-13 | 2010-10-13 | 广州和竺生物科技有限公司 | Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof |
| CN103045540A (en) * | 2011-10-14 | 2013-04-17 | 北京清美联创干细胞科技有限公司 | Novel method for promoting bone mesenchymal stem cells to be differentiated into neurons in rat brain |
| CN104561099A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | Preparation method of UCMSC (umbilical cord mesenchymal stem cell) modified with HGF (hepatocyte growth factor) gene and used for treating liver failure |
| CN105255836A (en) * | 2015-10-14 | 2016-01-20 | 紫程瑞生会(北京)生物技术发展有限公司 | Method for preparing person stem cells with improved neural restoration function and application of person stem cells |
| CN106110302A (en) * | 2016-07-28 | 2016-11-16 | 深圳爱生再生医学科技有限公司 | The stem cell medicine for the treatment of diabetic foot |
| CN108094404A (en) * | 2017-12-20 | 2018-06-01 | 北京臻惠康生物科技有限公司 | A kind of improved mesenchyme stem cell protection solution and application thereof |
-
2018
- 2018-06-21 CN CN201810645564.9A patent/CN108753730A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333542A (en) * | 2007-06-25 | 2008-12-31 | 何星儒 | Process for transplanting and repairing retina damnification in eye by gene modified mesenchyme stem cell |
| CN101857854A (en) * | 2009-04-13 | 2010-10-13 | 广州和竺生物科技有限公司 | Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof |
| CN103045540A (en) * | 2011-10-14 | 2013-04-17 | 北京清美联创干细胞科技有限公司 | Novel method for promoting bone mesenchymal stem cells to be differentiated into neurons in rat brain |
| CN104561099A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | Preparation method of UCMSC (umbilical cord mesenchymal stem cell) modified with HGF (hepatocyte growth factor) gene and used for treating liver failure |
| CN105255836A (en) * | 2015-10-14 | 2016-01-20 | 紫程瑞生会(北京)生物技术发展有限公司 | Method for preparing person stem cells with improved neural restoration function and application of person stem cells |
| CN106110302A (en) * | 2016-07-28 | 2016-11-16 | 深圳爱生再生医学科技有限公司 | The stem cell medicine for the treatment of diabetic foot |
| CN108094404A (en) * | 2017-12-20 | 2018-06-01 | 北京臻惠康生物科技有限公司 | A kind of improved mesenchyme stem cell protection solution and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| ANNA MACHALI´NSKA ET AL.: "Long-Term Neuroprotective Effects of NT-4–Engineered Mesenchymal Stem Cells Injected Intravitreally in a Mouse Model of Acute Retinal Injury", 《INVESTIGATIVE OPHTHALMOLOGY&VISUAL SCIENCE》 * |
| SUNG MIN AHN ET AL.: "Therapeutic Potential of a Combination of Electroacupuncture and TrkB-Expressing Mesenchymal Stem Cells for Ischemic Stroke", 《MOLECULAR NEUROBIOLOGY》 * |
| TSENG PT ET AL.: "ACCESSION NO:NM_006179,Homo sapiens neurotrophin 4 (NTF4), mRNA", 《GENBANK》 * |
| 侯宗柳 等: "《围产期成体干细胞基础与临床应用》", 31 October 2016 * |
| 孙兆明 等: "脑源性神经营养因子真核表达载体转染人脐带间充质干细胞", 《中国组织工程研究》 * |
| 朱榆红: "NT4-GFP转基因骨髓基质干细胞脑内移植对AD鼠行为、功能的影响", 《中国优秀博硕士学位论文全文数据库（博士） 医药卫生科技辑》 * |
| 陈宗道 等: "《食品生物技术概论》", 31 January 2008 * |
| 龙村: "《体外循环学》", 29 February 2004 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609554A (en) * | 2018-11-29 | 2019-04-12 | 南方医科大学 | A kind of umbilical cord mesenchymal stem cells and its preparation method and application of mir338 gene silencing |
| CN109609554B (en) * | 2018-11-29 | 2022-03-15 | 南方医科大学 | Cord mesenchymal stem cell with mir338 gene silencing function and preparation method and application thereof |
| CN111235115A (en) * | 2020-02-25 | 2020-06-05 | 南京鼓楼医院 | Recombinant mesenchymal stem cell, preparation method thereof and application thereof in preparing medicine for treating acute liver failure |
| CN113430231A (en) * | 2021-04-20 | 2021-09-24 | 华夏源细胞工程集团股份有限公司 | Method for efficiently infecting human umbilical cord mesenchymal stem cells by using pseudolentivirus vector |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2021097675A (en) | Aav vectors targeted to the central nervous system | |
| Nikkhah et al. | A microtransplantation approach for cell suspension grafting in the rat Parkinson model: a detailed account of the methodology | |
| US20060247195A1 (en) | Method of altering cell properties by administering rna | |
| RU2005105065A (en) | STEM ROOF CELLS AND METHODS FOR TREATING THEM WITH THE USE OF EYE DISEASES ASSOCIATED WITH VASCULAR NEW FORMATION | |
| AU2007299043B2 (en) | Expansion method for adult stem cells from blood, particularly peripheral blood, and relative application in medical field | |
| JP2004501113A (en) | Methods and formulations for controlled release of recombinant parvovirus vectors | |
| CN108753730A (en) | The umbilical cord mesenchymal stem cells and its construction method of a kind of NTF4 genetic modifications and application | |
| KR20170121340A (en) | Immortalized stem cells and medicinal composition and medicinal preparation comprising product thereof as active ingredient | |
| CN109689858A (en) | Method for generating mesoderm and/or endothelium colony forming cell like cell with body vessel Forming ability | |
| CN107970438A (en) | A kind of nerve regneration gel and its preparation method and application | |
| CN108588026A (en) | The preparation method and its usage of the clinical grade mescenchymal stem cell of height expression IL10 | |
| Wang et al. | Spinal cord injury target-immunotherapy with TNF-α autoregulated and feedback-controlled human umbilical cord mesenchymal stem cell derived exosomes remodelled by CRISPR/Cas9 plasmid | |
| US11622964B2 (en) | Method for destroying cellular mechanical homeostasis and promoting regeneration and repair of tissues and organs, and use thereof | |
| WO2003000871A1 (en) | Schwann cells originating in myeloid interstitial cells | |
| CN103865873B (en) | The allochthon of Subaerial blue green algae secretion and its application | |
| CN106011173B (en) | Preparation method of human oligodendrocyte progenitor cells for inhibiting nerve secondary injury, kit and application thereof | |
| CN105255836A (en) | Method for preparing person stem cells with improved neural restoration function and application of person stem cells | |
| CN110656087B (en) | MANF gene modified umbilical cord mesenchymal stem cell and preparation method and application thereof | |
| CN105924526B (en) | Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER1-NKT cell and its preparation method and application | |
| Zhang et al. | Bone marrow stromal cells transplantation promotes the resolution and recanalization of deep vein thrombosis in rabbits through regulating macrophage infiltration and angiogenesis | |
| CN108366567A (en) | Use the angiogenesis of stimulated placenta stem-cell | |
| CN105112368B (en) | A kind of stem cell medicine and its application method for treating retinosis | |
| CN104288781B (en) | Application in the medicine of preparation prevention and/or treatment ischemic ocular disease for the miR 329 | |
| CN113388586A (en) | Oncolytic virus NDV-NRP1 and construction method and application thereof | |
| TWI272107B (en) | A composition for gene therapy by gene transfer in vivo |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 226000 No. 1692 Xinghu Avenue, Nantong Development Zone, Jiangsu Province Applicant after: JIANGSU LUYISHENG BIOTECHNOLOGY Co.,Ltd. Address before: 226000 No. 1692 Xinghu Avenue, Nantong Development Zone, Jiangsu Province Applicant before: NANTONG LUYISHENG BIOTECHNOLOGY Co.,Ltd. |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181106 |