CN115369095A - Mouse auditory neuron immortalized cell line and construction method and application thereof - Google Patents
Mouse auditory neuron immortalized cell line and construction method and application thereof Download PDFInfo
- Publication number
- CN115369095A CN115369095A CN202211098985.7A CN202211098985A CN115369095A CN 115369095 A CN115369095 A CN 115369095A CN 202211098985 A CN202211098985 A CN 202211098985A CN 115369095 A CN115369095 A CN 115369095A
- Authority
- CN
- China
- Prior art keywords
- mouse
- cells
- cell line
- neuron
- immortalized cell
- 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
- 210000004027 cell Anatomy 0.000 title claims abstract description 86
- 210000002569 neuron Anatomy 0.000 title claims abstract description 57
- 238000010276 construction Methods 0.000 title claims abstract description 6
- 229950010131 puromycin Drugs 0.000 claims abstract description 15
- 210000001323 spiral ganglion Anatomy 0.000 claims abstract description 13
- 230000002018 overexpression Effects 0.000 claims abstract description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 18
- 206010011878 Deafness Diseases 0.000 claims description 15
- 231100000895 deafness Toxicity 0.000 claims description 13
- 208000016354 hearing loss disease Diseases 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 8
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 7
- 229960000723 ampicillin Drugs 0.000 claims description 7
- 239000012091 fetal bovine serum Substances 0.000 claims description 7
- 238000001890 transfection Methods 0.000 claims description 6
- 102000029816 Collagenase Human genes 0.000 claims description 5
- 108060005980 Collagenase Proteins 0.000 claims description 5
- 241000713666 Lentivirus Species 0.000 claims description 5
- 108010019160 Pancreatin Proteins 0.000 claims description 5
- 229960002424 collagenase Drugs 0.000 claims description 5
- 229940055695 pancreatin Drugs 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 230000029087 digestion Effects 0.000 claims description 4
- 238000010166 immunofluorescence Methods 0.000 claims description 4
- 238000003125 immunofluorescent labeling Methods 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 238000010171 animal model Methods 0.000 claims description 2
- 239000012502 diagnostic product Substances 0.000 claims description 2
- 239000003596 drug target Substances 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 1
- 210000003027 ear inner Anatomy 0.000 abstract description 9
- 238000011160 research Methods 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 6
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 abstract description 3
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 abstract description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 39
- 239000002609 medium Substances 0.000 description 9
- 241000283707 Capra Species 0.000 description 6
- 101001092197 Homo sapiens RNA binding protein fox-1 homolog 3 Proteins 0.000 description 5
- 102100035530 RNA binding protein fox-1 homolog 3 Human genes 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012737 fresh medium Substances 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 2
- 210000003477 cochlea Anatomy 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005485 electric heating Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 210000003625 skull Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010051542 Early Growth Response Protein 1 Proteins 0.000 description 1
- 102100023226 Early growth response protein 1 Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 206010029333 Neurosis Diseases 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 208000005400 Synovial Cyst Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 102000013814 Wnt Human genes 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000000609 ganglia Anatomy 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000002768 hair cell Anatomy 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 208000015238 neurotic disease Diseases 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229960005141 piperazine Drugs 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Images
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
- C12N5/0618—Cells of the nervous system
- C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2510/04—Immortalised cells
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Neurology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Acoustics & Sound (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Neurosurgery (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of immortalized cells, in particular to a mouse auditory neuron immortalized cell line and a construction method and application thereof. After the spiral ganglion cells of the inner ear of a mouse are separated, a SV40LT over-expression lentiviral vector (EF 1 alpha-SV 40-IRES-puromycin vector element) is used for transfecting the spiral ganglion cells, and cells which are successfully transfected are screened to obtain a mouse auditory neuron immortalized cell line SIO-SGN1 which has no obvious difference with the characters of the spiral ganglion cells of the inner ear of the primary mouse. And the immortalized cell line has simple in vitro culture conditions and high passage speed, and is a cell tool which can be stably used for auditory research.
Description
Technical Field
The invention relates to the technical field of immortalized cells, in particular to a mouse auditory neuron immortalized cell line and a construction method and application thereof.
Background
Deafness has become a major public health problem worldwide, and according to the statistics of the world health organization, about 15 hundred million people worldwide suffer from deafness disorders with different degrees, accounting for 19 percent of the world's general population. Deafness disorders have serious consequences for the family, society of the deaf and the deaf itself. The mechanism of occurrence of deafness is complex and is susceptible to various factors such as environment, drugs, age, etc., so the research on the mechanism of occurrence of deafness and the prevention and treatment methods still needs to be further advanced.
Spiral ganglion cells, which are auditory neurons in the inner ear, are the first-order neurons on the auditory conduction pathway and are capable of converting acoustic signals from hair cells into electrical signals and conducting them up to the auditory centers of the brain, thus forming the sense of hearing. Therefore, the spiral ganglion neurons play a very important role in the process of auditory conduction, and are also the key subjects of current auditory research. However, since there are numerous cell types In the inner ear, it is difficult to obtain Purified Spiral Ganglion cells by common isolation Culture methods, and the Spiral Ganglion neurons belong to terminally differentiated cells, are difficult to passage during In vitro Culture, and have strict requirements on Culture environments and limited Survival times when cultured In vitro, such as In the literature Wei, D.et. Al., survival, synaptogenesis, and regeneration of adult motor Spiral growth Neuron navigation Journal of Neurobiology, 2007.67 (1): p.108-122 and Yan, W.et. Al., A Three-Dimensional Current System with matrix proteins purification Purified Spiral Neuron navigation simulation and mutation In vitro navigation Neurol, 8.55 (p.2013): 2070-2084, and thus have limited applications In studies thereof. Immortalization techniques are required to establish immortalized mouse auditory neuron cell lines.
Disclosure of Invention
Aiming at the problems in the prior art, the inventor researches the immortalized mouse inner ear auditory ganglion cell, after separating the mouse inner ear spiral ganglion cell, the SV40LT overexpression lentivirus vector containing EF1 alpha-SV 40-IRES-puromycin vector element is used for transfecting spiral ganglion, and the successfully transfected cell is screened, so that the mouse auditory neuron immortalized cell line SIO-SGN1 with no obvious difference with the primary mouse inner ear spiral ganglion cell character is obtained, and the immortalized cell line can be stably subcultured, in vitro culture and passage of the inner ear spiral ganglion cell are realized, thereby providing convenient conditions for auditory research. And the immortalized cell line has simple in vitro culture conditions and high passage speed, and is a cell tool which can be stably used for auditory research.
Firstly, the mouse auditory neuron immortalized cell line has the preservation number of CCTCC NO: C202275.
the mouse auditory neuron immortal cell line is constructed and obtained by the following method, and the specific process is as follows:
(1) Isolation and culture of C57 mouse auditory neurons: taking a C57 mouse born for 1d to dissect a cochlear, carrying out enzymolysis and digestion, suspending single cells, and then, laying a bottle for culture;
(2) And (3) carrying out immunofluorescence identification on the separated cells: performing immunofluorescence staining after cell slide, and observing the staining condition of the neuron marker;
(3) C57 mouse auditory neuron cells were transfected with SV40LT overexpressing lentiviruses: the helical ganglia were transfected with the SV40LT overexpression lentiviral vector (EF 1. Alpha. -SV40-IRES-puromycin vector element). After transfection, cells are screened after being amplified for 3-4 generations;
(4) Screening of C57 mouse auditory neuron cells: cells successfully transfected were selected using puromycin.
Aiming at the mouse auditory neuron immortalized cell line obtained by the technical scheme of the invention, the invention further provides a culture medium and a culture method suitable for the amplification culture of the mouse auditory neuron immortalized cell line, wherein the culture medium is specifically 1640 culture medium containing 5 to 10 percent of fetal bovine serum and 1 mu L/ml of ampicillin, the culture medium is placed at 37 ℃ and contains 5 percent of CO 2 The culture is carried out in a constant temperature incubator.
Preferably, the enzymolysis in the step (1) adopts pancreatin and collagenase type I; most preferably, 0.25% pancreatin and 0.1% collagenase type I are used.
The mouse auditory neuron immortalized cell line can be used for the following purposes:
(1) For preparing an auditory neuron cell animal model;
(2) Used for developing deafness drug targets;
(3) Used for preparing deafness diagnostic products;
(4) Used for developing or/and screening or/and evaluating deafness treatment drugs.
In addition, the mouse auditory neuron immortalized cell line can also be used for other deafness related researches, preparation of deafness related products and the like.
The method for constructing the immortalized mouse auditory neuron cell line provided by the invention separates and obtains normal auditory neuron cells from the cochlea of the suckling mouse, carries out transfection by SV40LT over-expressed slow virus, and obtains the immortalized mouse auditory neuron cell line after successful transfection. The SV40LT overexpression lentiviral vector containing the EF1 alpha-SV 40-IRES-puromycin vector element is adopted to transfect the spiral ganglion, and the method has the obvious advantages of capability of infecting terminal differentiated cells, high transfection efficiency and the like. After twenty passages, the immortalized auditory neuron cell line has no obvious difference in form compared with the first passage cell line, has good cell growth state, firm adherence and clear outline, and expresses the same marker as the primary auditory neuron cell.
Preservation information
Preservation time: 26/04/2022;
the name of the depository: china center for type culture Collection;
the preservation number is as follows: CCTCC NO: c202275;
the address of the depository: wuhan university in Wuhan, china;
and (3) classification and naming: mouse auditory neurons immortalized cell line SIO-SGN1.
Drawings
FIG. 1 is a diagram of the identification of mouse auditory neuron immortalized cell lines NeuN and Tuj1 by antibody immunofluorescence staining.
FIG. 2 is a cell morphology diagram of C57 mouse auditory neurons isolated in example 1 after cell plating.
FIG. 3 is a diagram showing immunofluorescence assay of Tuj1 antibody as an isolated cell of C57 mouse auditory neuron cell in example 1.
FIG. 4 is a comparison graph of cell morphology of the first, tenth and twentieth generations of the mouse auditory neuron immortalized cell line of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the present invention is limited to the examples. The techniques realized based on the above contents of the present invention all belong to the scope of the present invention, and the following embodiments are all completed by using the conventional prior art except for the specific description.
The following examples are provided by the prior art, using the following instruments and reagents.
(1) Instrument (Instrument name: specification, manufacturer):
the biological safety cabinet: BSC-1500 IIA 2-X, N.J. Beixi Biotechnology, inc., jinan;
CO2 cell incubator: BC-J160S, shanghai Boxun industries, inc.;
fluorescence inverted microscope: DS-Ri2, nikon;
a high-speed refrigerated centrifuge: multifuge X1R, thermo Fisher;
electric heating constant temperature air blast drying cabinet: DHG-9123A, shanghai essence macro laboratory equipment ltd;
electric heating constant temperature oscillation water tank: DK-2B, shanghai sperm macro laboratory Equipment Co., ltd.
(2) Reagent consumables (reagent name: specification/goods number, manufacturer):
t25 cell culture flask: 430639, corning;
24-well plate special cell slide: YA0350, cammed;
cell culture well plate: WHB-24, shanghai lying Macro Biotechnology, inc.;
fetal bovine serum: 1414426, gibco;
0.25% trypsin (containing 0.02% edta): 1734858, gibco;
collagenase i (Collagenase i): 17018029, gibco;
paraformaldehyde (PFA): p1110, solarbio;
DAPI：C0060，Solarbio；
Triton X-100：T8200，Solarbio；
goat serum: SL038, carmeder;
tuj1:66375-1-AP, camarded;
NeuN：12943，Cell signaling pathway；
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488：A-11029，Thermo fisher；
CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L): SA00013-4, cammede;
ampicillin: a1170, gibco;
fluorocount-G fluorescent encapsulated tablet: 0100-01, cammed;
SV40 overexpresses lentiviruses: and (4) Cammede.
Example 1 mouse auditory neuron immortalized cell line construction
(1) Isolation and culture of C57 mouse auditory neurons:
1) Soaking C57 mice born for 1d in 75% alcohol for sterilization;
2) Rapidly cutting off the head of the disinfected C57 mouse, cutting the skull along the macropore of the occiput of the mouse, and removing the skull and brain tissues;
3) Fully exposing the cochlea under a dissecting microscope, opening the volute, completely taking out the semi-membrane labyrinth and the volute, peeling off the outer side wall, and tearing off nerve fiber tissues in the basement membrane and the volute as much as possible;
4) Cutting the rest tissue blocks, adding 0.25% pancreatin and 0.1% I-type collagenase, digesting in a shaking water bath at 37 deg.C for 8min (5-10 min), and preferably taking out and lightly beating for 3-5 min to make digestion more complete;
5) Stopping digestion with serum, gently blowing, and filtering with 100 μm filter screen; collecting 300g (200-400 g) of digested tissue, and centrifuging for 5min (5-8 min);
6) After centrifugation, the pellet was resuspended in 1640 medium containing 10% fetal bovine serum and 1. Mu.L/ml ampicillin, and cultured in flasks, and the morphology of the cells after flasks was as shown in FIG. 2.
(2) And (3) carrying out immunofluorescence identification on the separated cells:
1) Cell slide: putting 3 glass sheets into 24-well plate, adding 1640 culture medium containing 10% fetal calf serum and 1 μ L/mL ampicillin into each well for 1mL, adding 0.02 milli-cells/well, and placing in incubator for 2h (or overnight);
2) Fixing: after cell slide, sucking out the culture medium, washing with PBS for 1 time, adding 4% PFA, fixing at 4 deg.C for 30min, washing with PBS for 3 × 5 min/time (or standing at 4 deg.C overnight without sucking out PBS for the last time);
3) Membrane rupture and sealing: removing water from the slide, placing the slide on a culture dish support, taking 50uL of membrane-breaking sealing solution (0.5% Trition X-100 is mixed with PBS 1, then 10% serum is added) and dripping the solution on a waterproof membrane, and covering one surface of the slide with cells for 2h;
4) Primary antibody incubation:
preparing a primary antibody: diluting Tuj1 antibody with PBS 1 to prepare primary anti-dilution solution;
after the membrane is broken and sealed, 50uL of primary anti-dilution solution is taken to be put on a waterproof membrane (in a wet box), a slide (the side with cells) is covered and placed at 4 ℃ overnight (generally 12-24 hours);
5) And (3) secondary antibody incubation: discarding the primary antibody incubation solution, diluting a Goat Anti-Rabbit IgG (H + L) fluorescent secondary antibody and PBS according to a ratio of 1;
6) Embedding: the staining results of 1 drop of fluorocount-G fluorescent blocking tablet on each slide, covering the side with cells, are shown in FIG. 3.
(3) C57 mouse auditory neuron cells were transfected with SV40LT overexpressing lentiviruses:
1) C57 mouse auditory neuron cells were seeded in 6-well plates at about 1X 10 cells per well 5 Is (also can be 0.5-2X 10) 5 Any number of cells), the next day, after the cells adhere to the wall, changing the liquid;
3) 1mL of 1640 medium containing 10% fetal bovine serum and 1. Mu.L/mL ampicillin was added to each well, 20. Mu.L (any value in the range of 10 to 30. Mu.L can be selected) of SV40LT over-expression lentiviral vector (EF 1. Alpha. -SV40-IRES-puromycin vector element) was added thereto, the culture was continued after mixing, the state of the cells was observed after 12 hours, and the medium was replaced with fresh medium, and when the cells were grown to the bottom of the plate, they were passaged to a T25 flask.
(4) Screening of C57 mouse auditory neuron cells: after transfection, cells are screened after 3-4 generations of amplification, and the specific steps are as follows:
1) Determination of the kill curve: untransfected C57 mouse auditory neuron cells were plated in 24 well plates at a density of 0.05million per well (or any density in the range of 0.01-0.1 million), incubated overnight, the next day, old medium was removed from the 24 well plates, fresh medium containing different concentrations of puromycin (1 ug/mL,2ug/mL,3ug/mL,4ug/mL,5 ug/mL,6ug/mL,7 ug/mL) was added to the 24 well plates that had been plated with cells, fresh selection medium was replaced every 2 days while observing the survival rate of cells daily to determine the concentration and duration of action of puromycin. The minimum puromycin concentration used was the lowest screening concentration that killed all cells within 1-4 days from puromycin screening. The final result is: the using concentration of puromycin is 2ug/mL, and the action time is 2d;
2) Puromycin selection of transfected cells: on the first day, transfected C57 mouse auditory neuron cells were plated in 24-well plates at a density of 0.05million (or any value from 0.01 to 0.1 million) per well and incubated overnight; the next day, the old medium in the 24-well plate was removed, the selection medium containing puromycin (2 ug/mL) was added, incubation was performed, the selection medium was replaced with fresh one every 2 days and the survival rate of the cells was observed, and the surviving cells were C57 mouse auditory neuron cells that were successfully transfected.
Example 2 mouse auditory neurons immortalized cell lines expansion culture
The mouse auditory neuron immortalized cell line obtained in example 1 was amplified and cultured in 1640 medium containing 10% fetal bovine serum (any value in the range of 5% to 10% fetal bovine serum can be selected) and 1. Mu.L/ml ampicillin,placing at 37 deg.C, with 5% CO 2 The culture was carried out in a constant temperature incubator, and the fresh medium was changed every 2 days. Under the culture conditions, the cells proliferated rapidly, and after passage to the tenth generation and the twentieth generation, the cells did not change significantly compared with the first generation, and the cell morphology is shown in fig. 4.
Example 3 identification of mouse auditory neuron immortalized cell lines
The mouse auditory neuron immortalized cell line prepared in example 1 was detected by immunofluorescence staining, which was immunostained using antibodies to the neuronal markers NeuN, tuj1, wherein
Experimental group 1: the primary antibody adopts Tuj1 antibody, and the secondary antibody adopts Goat anti-Mouse IgG (H + L) antibody;
experimental group 2: the primary antibody was NeuN antibody, and the secondary antibody was Goat Anti-Rabbit IgG (H + L) antibody.
Reference is made to the published documents Wang, M., et al, characterisation of EGR-1 Expression in the Audio coding Following Kanamycin-Induced heating Loss in Mice.J. Mol Neurosis, 2021.71 (11): p.2260-2274. Or Liu, W., et al, wnt signalling activities TP53-Induced diabetes and Apoptosis Regulator and Protect Agents analysis Cisplatin-Induced Spiral gain nerve in the Motor coding residue in Anxied Redox Signal 2019.30 (11): p.1389-1410.
Experimental identification results show that the immortalized cell line expresses the neuron markers Tuj1 and NeuN, and the neuron characteristics of the cell line are verified, as shown in figure 1.
Claims (4)
1. An immortalized cell line of mouse auditory neurons, which has a preservation number of CCTCC NO: C202275.
2. the method for constructing an immortalized cell line of mouse auditory neurons according to claim 1, wherein the method comprises the following steps:
(1) Isolation and culture of C57 mouse auditory neurons: taking a C57 mouse born for 1d to dissect a cochlear, carrying out enzymolysis and digestion, suspending single cells, and then, laying a bottle for culture;
(2) And (3) carrying out immunofluorescence identification on the separated cells: performing immunofluorescence staining after cell slide, and observing the staining condition of the neuron marker;
(3) C57 mouse auditory neuron cells were transfected with SV40LT overexpressing lentiviruses: transfecting a spiral ganglion by using an SV40LT overexpression lentiviral vector element, namely an EF1 alpha-SV 40-IRES-puromycin vector element; after transfection, cells are screened after being amplified for 3-4 generations;
(4) Screening of C57 mouse auditory neuron cells: screening cells successfully transfected by puromycin;
pancreatin and I-type collagenase are adopted for enzymolysis in the step (1);
the culture medium adopted in the construction process is 1640 culture medium containing 5% -10% fetal bovine serum and 1 uL/ml ampicillin.
3. The method for constructing an immortalized cell line of mouse auditory neurons according to claim 2, wherein the enzymatic hydrolysis in step (1) is performed using 0.25% pancreatin and 0.1% collagenase type i.
4. The use of the mouse auditory neuron immortalized cell line of claim 1, which is any one or more of the following:
(1) For preparing an auditory neuron cell animal model;
(2) The method is used for developing deafness drug targets;
(3) For preparing deafness diagnostic products;
(4) Used for developing or/and screening or/and evaluating deafness treatment drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211098985.7A CN115369095A (en) | 2022-09-09 | 2022-09-09 | Mouse auditory neuron immortalized cell line and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211098985.7A CN115369095A (en) | 2022-09-09 | 2022-09-09 | Mouse auditory neuron immortalized cell line and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115369095A true CN115369095A (en) | 2022-11-22 |
Family
ID=84071988
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211098985.7A Pending CN115369095A (en) | 2022-09-09 | 2022-09-09 | Mouse auditory neuron immortalized cell line and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115369095A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215577A (en) * | 2008-01-14 | 2008-07-09 | 中国海洋大学 | General purpose method for preparing integration type cell immortalization vector |
| CN102229912A (en) * | 2011-05-18 | 2011-11-02 | 中国人民解放军总医院 | Cochlear greater epithelial ridge (GER) cell line and its application |
| CN110904051A (en) * | 2019-12-26 | 2020-03-24 | 上海交通大学医学院附属第九人民医院 | Human auditory neuroma immortalized cell line, preparation method and application thereof |
| CN111454990A (en) * | 2019-11-25 | 2020-07-28 | 上海交通大学医学院附属第九人民医院 | Human jugular auxiliary nerve ganglionic tumor immortalized cell strain and application thereof |
| CN112608878A (en) * | 2020-12-18 | 2021-04-06 | 复旦大学附属眼耳鼻喉科医院 | In-vitro cochlear micro-organ functional unit and three-dimensional construction method and application thereof |
| CN112920998A (en) * | 2021-02-05 | 2021-06-08 | 复旦大学附属眼耳鼻喉科医院 | Establishment method and application of cochlear body culture system of adult mouse |
| CN114807043A (en) * | 2022-03-24 | 2022-07-29 | 上海交通大学医学院附属第九人民医院 | Human-derived vestibular schlemma immortalized cell line and construction method thereof |
-
2022
- 2022-09-09 CN CN202211098985.7A patent/CN115369095A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215577A (en) * | 2008-01-14 | 2008-07-09 | 中国海洋大学 | General purpose method for preparing integration type cell immortalization vector |
| CN102229912A (en) * | 2011-05-18 | 2011-11-02 | 中国人民解放军总医院 | Cochlear greater epithelial ridge (GER) cell line and its application |
| CN111454990A (en) * | 2019-11-25 | 2020-07-28 | 上海交通大学医学院附属第九人民医院 | Human jugular auxiliary nerve ganglionic tumor immortalized cell strain and application thereof |
| CN110904051A (en) * | 2019-12-26 | 2020-03-24 | 上海交通大学医学院附属第九人民医院 | Human auditory neuroma immortalized cell line, preparation method and application thereof |
| CN112608878A (en) * | 2020-12-18 | 2021-04-06 | 复旦大学附属眼耳鼻喉科医院 | In-vitro cochlear micro-organ functional unit and three-dimensional construction method and application thereof |
| CN112920998A (en) * | 2021-02-05 | 2021-06-08 | 复旦大学附属眼耳鼻喉科医院 | Establishment method and application of cochlear body culture system of adult mouse |
| CN114807043A (en) * | 2022-03-24 | 2022-07-29 | 上海交通大学医学院附属第九人民医院 | Human-derived vestibular schlemma immortalized cell line and construction method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 张媛等: "大鼠耳蜗大上皮嵴及小上皮嵴细胞永生细胞系的建立", 临床耳鼻咽喉科杂志, vol. 20, no. 10, 31 May 2006 (2006-05-31), pages 460 - 462 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ragnarsson et al. | Basic biological sciences isolation and growth of human periodontal ligament cells in vitro | |
| KR100519227B1 (en) | A method for transdifferentiating mesenchymal stem cells into neuronal cells | |
| JP4365406B2 (en) | How to make teeth from bone marrow cells | |
| CN110904051B (en) | Human auditory neuroma immortalized cell line, preparation method and application thereof | |
| US20220154139A1 (en) | YAP1 Gene-Modified Mesenchymal Stem Cell and Preparation Method Thereof | |
| Lorenzo et al. | Generation of mouse and human induced pluripotent stem cells (iPSC) from primary somatic cells | |
| EA028902B1 (en) | Method of generating induced pluripotent stem cells and differentiated cells | |
| CN101351118B (en) | Use of apoptotic cells ex vivo to generate regulatory t cells | |
| CN111467373A (en) | Dental pulp stem cell exosome preparation, preparation method and application thereof | |
| CN109706180A (en) | A kind of umbilical cord mesenchymal stem cells, which are overexpressed IDO, enhances immunosuppressive method and application | |
| CN108251378B (en) | A kind of interstital stem cell excretion body and its preparation method and application being overexpressed PTGDS gene | |
| CN109402062A (en) | Application of the ZIP1 gene in the product that preparation inhibits apoptosis of mesenchymal stem cell | |
| CN114807043A (en) | Human-derived vestibular schlemma immortalized cell line and construction method thereof | |
| Zhu et al. | Isolation and Long‐Term Expansion of Functional, Myelinating Oligodendrocyte Progenitor Cells from Neonatal Rat Brain | |
| CN107779429A (en) | A kind of tissue-derived fibroblast quick separating cultural method of application on human skin | |
| AU725173B2 (en) | Immortalized retinal cell lines and their applications | |
| CN112608878B (en) | In-vitro cochlear micro-organ functional unit and three-dimensional construction method and application thereof | |
| CN115369095A (en) | Mouse auditory neuron immortalized cell line and construction method and application thereof | |
| Weinstein et al. | Isolation and purification of primary Schwann cells | |
| CN113025661A (en) | Construction method of immortalized musk glandular epithelial cells | |
| Gong | Culture of mouse olfactory sensory neurons | |
| CN113817777B (en) | Congenital giant black nevus benign tumor cell line from human and construction method thereof | |
| US20040259249A1 (en) | Method of making stem cells from differentiated cells | |
| CN107460166A (en) | The isolated culture method of one breeder GHR depletion mutant sarcoblasts | |
| Lemus et al. | Odontogenesis and amelogenesis in interacting lizard—Quail tissue combinations |
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 |