CN108070567A - A kind of urine derived cell strain of immortalization and its construction method - Google Patents
A kind of urine derived cell strain of immortalization and its construction method Download PDFInfo
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- CN108070567A CN108070567A CN201810052365.7A CN201810052365A CN108070567A CN 108070567 A CN108070567 A CN 108070567A CN 201810052365 A CN201810052365 A CN 201810052365A CN 108070567 A CN108070567 A CN 108070567A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0684—Cells of the urinary tract or kidneys
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- C12N2510/00—Genetically modified cells
- C12N2510/04—Immortalised cells
Abstract
The strain of urine derived cell and its construction method the invention discloses a kind of immortalization, belong to biological technical field.The urine derived cell strain of the immortalization is will to carry the Transfected Recombinant Plasmid of immutalizing gene or DNA fragmentation into obtained by urine derived cell.SV40LT Transfected Recombinant Plasmids into urine derived cell, are constructed the urine derived cell strain of immortalization by applicant.And by comparing, the urine derived cell strain that safer reliable gene editing method is immortalized preferably is gone out.When the urine derived cell strain culture of the immortalization is to 35 generation, cellular morphology is very young, and still without apparent aging phenomenon；Cell almost increases in logarithm, is 3 times cultivated to the 4th generation primary urine derived cell growth rate.The urine derived cell strain indices that identification and analysis display immortalizes belong to normal.It recovers after freezing 1 month, adherence rate is up to 95%, growth and passage that can be steady in a long-term.
Description
Technical field
The invention belongs to biological technical fields, and in particular to a kind of urine derived cell strain of immortalization and its structure side
Method.
Background technology
Existing numerous studies show influence of the contemporary people because of eating habit and social environment, and the urinary tract system is (including wing
Guang and kidney) there are a variety of diseases, renal-related conditions such as kidney chronic glomerulonephritis, pyelonephritis, nephrosis end simulator sickness, acute
Kidney failure, high blood nephrosis etc. and bladder can be because the factors such as Exposed touch the chemical substances such as benzidine and cause bladder
Tumour, etc..Scientific research personnel carries out these the urinary tract systems the research of relevant disease, is typically taken by biopsy
The method of sample obtains the object of research, this behavior for having invasive donor bladder or kidney can be caused relevant wound,
Local organization bleeding, infection or donor are uncomfortable, this also resistance for causing patient more.And the cell being collected into from urine equally comes
Renal plevis, ureter, bladder, anterior urethra scaly epithelium are come from, it can be as the extracorporeal model system of a replacement, for the urinary tract
The research of system related disorders.In addition, urine derived cell is to collect culture from urine to obtain, mode of operation is relatively simple,
Without any traumatic.Therefore, urine derived cell is the good external model of the urinary tract systemic disease.Scientific research personnel can pass through
Urine derived cell model studies relevant the urinary tract systemic disease, therefore collects the urine derived cell of different donors
It is quite important to building different urine derived cell models and related the urinary tract systemic disease research.Urine derived cell is a kind of
Primary cell by collecting donor urine and being separately cultured to obtain, however is collected from donor urine and cultivates to obtain
The success or not of urine derived cell is restricted by many factors, such as the volume of urine specimen, pH value, osmotic pressure, urine donor
The factors such as amylase activity and time for being retained with urine in bladder in oxalic acid content, urine in age, urine, if wherein
One is not reaching to optimum state, can all influence the result that urine derived cell collects culture.R.Belik, W et al. once reported from
0-6300 urine derived cell, the urine derived cell that wherein most sample collection arrives only are collected in the urine of 1L
Amount is less than 1000, and the urine derived cell collected in urine specimen and the urine specimen amount that survives only account for institute
Have the 37% of urine specimen amount.In addition, even there is appropriate number of urine derived cell in sample, in incubation there is also
It many factors and restricts obtaining for urine derived cell.Different from other conventional primary cells, urine derived cell and non-sourcing
In tissue or organ, because its radix is small, the cell concentration needed for us need to can be obtained through multiple external division growth.But thin
In the case that cell concentration is few when born of the same parents cultivate early stage, urine derived cell can be in the incubation period of long period, and more difficult to get access right
Number growth periods, thus cell easily occur apoptosis and it is dead phenomena such as.And urine derived cell is in itself as a kind of primary thin
Born of the same parents, subculture number is very limited in vitro, and cell is with regard to gradual aging and can not generally when 6 generation to 8 generation
It is further continued for passage to use, this easily influences its research in terms of the disease of the urinary tract system, medicine sieve and diagnosis.Therefore, except
Outside being studied in primary urine derived cell level, build the urine derived cell strain of immortalization can solve energetically it is primary
Urine derived cell in vitro can not long-term cultivation the problem of, and can from cellular level further studying uropoiesis road system
Relevant disease.
Foreign gene is mostly imported into cell using virus as the carrier of gene transfer at present, although viral vectors by into
Row repeatedly transformation, virulent gene therein has been removed, but there are still potential secondary toxic action when expressing in vivo, it is such as prominent
The wild-type virus of change.In addition, viral multigelation can cause virus titer to reduce, therefore infected all using virus every time
Viral packaging need to be re-started, so as to cause the difference between batch of cell in cell bank, and anti-virus operation need to be other in higher security level
Under the conditions of carry out.Nearly ten years, using genetic engineering transformation artificial nuclease appearance, realize more efficient and more accurately
The efficiency of nonhomologous end reparation and homologous recombination greatly improved in gene editing, and new machine is brought for the genetic modification of species
It meets.Gene editing technology includes CRISPR-Cas9, TALEN, ZFN, CRISPR-Cpf1, other cause DNA break and insertion is outer
The gene editing means of source DNA, without DNA break homologous recombination, locus specificity recombinate etc. gene editings method, moreover, gene
Editing technique has the characteristics that at low cost, design is simple, easy to operate, experimental period is short, uses it for immortalized cell line structure
During accurate fixed point transformation can be carried out to genome in minimum zone, farthest avoid such as using it is viral as
To canceration caused by cell possibility during the carrier progress immortalized cells structure of gene transfer.Therefore, gene editing technology is utilized
The structure of the urine derived cell strain immortalized can retain the biological characteristics of primary urine derived cell to the greatest extent.
Biological sample bank is known as biobank (Biobank), is primarily referred to as standardization and collects, handles, storing and using strong
The large biological molecule of health or disease organism, cell, tissue and organ equal samples are (including human organ tissue, whole blood, blood plasma
Deng) and data and its quality control, the information such as clinic relevant with these biological samples, pathology, treatment, follow-up, informed consent
Management and application system.Biological sample bank includes polytype, except common tissue, organ storehouse, as blood storehouse, cornea storehouse,
Bone MarrowBank etc. further includes the cell line (being) of normal cell, genetic mutation cell, tumour cell and hybridoma cell strain (being)
Storehouse, these biological sample banks (Biobank) grind for the major diseases such as blood disease, disease of immune system, diabetes, malignant tumour
Study carefully and play very important impetus.For European countries, the biological sample bank in China is not also very perfect, has
It waits to perfect.And the foundation in the relevant cell storehouse there is presently no the urine derived cell strain of immortalization, and urine derived cell
Acquisition has the characteristics that the easier mode of more other cells and to donor hurtless measure, therefore, builds a urine derived cell
The cell bank of strain simultaneously carries out the cell of storage corresponding STR partings and HLA partings, establishes the cellular informatics archives for detection,
That is urine derived cell storehouse, it is particularly significant to the construction of improving China's biological sample bank.The foundation in urine derived cell storehouse can incite somebody to action
The DNA fossil datas of people, hereditary information system, the information of human body gene type monopolizing characteristic are stored, and can be human diseases
Research provides the sample of high quality.It can be the gene status of analysis relevant disease, analyze its reason and mechanism, be such as cancer, the heart
Control, prevention and the clinical treatment of the diseases such as popular name for, diabetes and senile dementia provide scientific basis.In addition, during the nearly last ten years,
Before genomics, functional genomics, high flux biochip, new-generation sequencing and bioinformatics, high throughput RNA etc.
Along the fast development of technology, urine derived cell storehouse is alternatively arranged as the important tool of translational medicine research, it can be not only base
Plinth research provides research object entity sample, the data message of specification, and can be various drugs, diagnosis, biological products, guarantor
The research and development of the products such as strong product, cosmetics provide reliable biological sample, data message and research and application service etc..
Therefore in conclusion the urine derived cell storehouse for establishing an immortalization is very necessary, it may have very great
Meaning.
The content of the invention
The strain of urine derived cell and its construction method and application it is an object of the invention to provide a kind of immortalization.
The technical solution used in the present invention is：
A kind of urine derived cell strain of immortalization, the cell line derive from urine specimen, possess external infinite multiplication
Or the ability close to infinite multiplication.
Preferably, the urine derived cell strain of the immortalization is the importing external source immutalizing gene in urine derived cell
Or DNA segment is built-up, the immutalizing gene or DNA segment include SV40LT, Bmi1, E6, E7, p53 mutant, Myc,
C-Jun and Mdm2 is at least one.
Preferably, the immutalizing gene is SV40LT or Myc.
Since SV40LT is the section of DNA sequence in SV40 viral genomes source, stable integration occurs with host cell DNA
Long-term existence is in intracellular afterwards, may change the primary characteristic of primary cell, and potentially dangerous property.The present invention is to existing
Some be transferred to method and screened and studied, the method for wherein gene editing is optimal, can be well by immutalizing gene
SV40LT is transferred to urine derived cell, stablizes 35 generations of passage, while can keep the normal karyotype of urine derived cell.
A kind of construction method of the urine derived cell strain of immortalization, comprises the following steps：Immutalizing gene will be carried
Or the Transfected Recombinant Plasmid of DNA fragmentation is into the urine derived cell strain that immortalization is built into urine derived cell.
Preferably, the immutalizing gene or DNA fragmentation include SV40LT, Bmi1, E6, E7, p53 mutant, Myc, c-
At least one of Jun and Mdm2.
Preferably, the immutalizing gene is SV40LT or Myc.
Preferably, the Transfected Recombinant Plasmid of immutalizing gene or DNA fragmentation will be carried into the method for urine derived cell
Include the use of at least one of retroviral vector, slow virus carrier, gene editing (i.e. " fixed point gene knock-in ").
Preferably, the urine derived cell immortalized using gene editing structure：SgRNA is found out, sgRNA is building up to
After gRNA Cloning Vector (Plasmid 41824, Adegene) plasmid, then it is transformed into DH5 α competent cells, picking
Monoclonal bacterial strain do bacterium colony PCR, PCR the result positive inoculation it is small carry after send sequencing, correct bacterial strain is sequenced and does plasmid again
In carry, by the method for restructuring, SV40LT genes are inserted into inside Donor plasmids, NruI digestions processing, recycle SV40LT
Donor segments obtain SV40LT recombinant plasmids.SV40LT recombinant plasmids are imported into primary urine source by the method for electroporation
Cell interior, so as to being immortalized urine derived cell strain.Applicant utilizes the side of gene editing (CRISPR) by verification
Method by immutalizing gene SV40LT recombinate into urine derived cell and the urine derived cell strain for being immortalized be it is optimal,
35 generations of passage can be stablized, while the normal karyotype of urine derived cell can be kept.
Preferably, target sgRNA special in gene editing is as follows：
sgRNA-AAVS1-1：5’TATAAGGTGGTCCCAGCTCG 3’(SEQ ID NO：1)；
sgRNA-AAVS1-2：5’AGGGCCGGTTAATGTGGCTC 3’(SEQ ID NO：2).
Preferably, the accurate cutting DNA of Cas9 nucleases is guided by sgRNA-AAVS1 during gene editing.AAVS1 is
One by verifying, can ensure that the safe site for being transferred to DNA fragmentation expectation function.The site is an open chromosome knob
Structure can guarantee that is turned enters gene by normal transcription.Target the CRISPR-Cas9 system energy specific cleavages mankind the in AAVS1 sites
AAVS1 sites on No. 19 chromosomes, generation DNA double chain are broken, and trigger the natural repair mechanism of DNA, inductive site and AAVS1
Homologous recombination occurs between donor dna clone, the safe site DNA fragmentation on donor clone being integrated on genome.
Preferably, Cas9 nucleases are expressed by Cas9-D10A expression plasmids during gene editing.Cas9-D10A is cut
Mouth enzyme cooperation a pair of sgRNA is used, it is possible to reduce unexpected off-target gene modification.It is produced in two opposite DNA chain respectively
Raw notch, so as to form functional double-strand break.This design can reduce undershooting-effect to greatest extent, at the same keep efficiently and
Special genetic modification again.
Preferably, damaged during gene editing by Donor recombinant fragments in DNA break position DNA plerosis.Donor
Target fragment more accurately can be inserted into safe site by recombinant fragment.
Preferably, the urine derived cell strain immortalized using slow virus carrier structure：By round pcr from 293T/17
SV40LT genetic fragments are obtained in cytogene, are carried SV40LT genetic fragments and pSin-EF2 with the method for gene recombination technology
Constitution burl closes, and forms pSin-EF2-SV40LT, digestion identification recombinant plasmid.By the method for recombinant plasmid calcium phosphate transfection
It imports inside urine derived cell, so as to the urine derived cell strain for being immortalized.
Preferably, the urine derived cell strain immortalized using retroviral vector construct：By round pcr from
SV40LT genetic fragments are obtained in 293T/17 cytogenes, with the method for gene recombination technology by SV40LT genetic fragments with
PBabe-puro vector plasmids combine, and form pBabe-puro-SV40LT, digestion identification recombinant plasmid.By recombinant plasmid phosphorus
The method of sour calcium transfection is imported inside urine derived cell, so as to the urine derived cell strain for being immortalized.
The urine derived cell strain that is immortalized described in any of the above-described is built in the urine derived cell strain of immortalization in storehouse
Application.
The beneficial effects of the invention are as follows：
Present invention applicant by the Transfected Recombinant Plasmid containing immutalizing gene or DNA fragmentation into urine derived cell,
The urine derived cell strain of immortalization is constructed, and storehouse is built applied to the urine derived cell strain immortalized.
Applicant by comparing, preferably goes out safer reliable gene editing mode and transfects immutalizing gene SV40LT
The urine derived cell strain GE-HUC immortalized into urine derived cell.The urine derived cell strain that the present invention immortalizes
When GE-HUC cultures are to 35 generation, cellular morphology is very young, and still without apparent aging phenomenon.And primary urine derived cell culture
During to 4 generation, cellular morphology apparent aging.The urine derived cell strain GE-HUC that present invention culture is immortalized to the 35th generation
Almost increase in logarithm, be 3 times cultivated to the 4th generation primary urine derived cell growth rate.It is identified by caryogram, oncogenicity
The analyses such as detection find, the urine derived cell strain GE-HUC indices of immortalization that the present invention is obtained by gene editing mode
Belong to normal.After being frozen, recovery culture is carried out after 1 month, adherent rate is up to 95% after cell recovery, can be long-term
Stable growth and passage.
Description of the drawings
Fig. 1 collects incubation aspect graph for primary urine derived cell；
Fig. 2 is cellular morphology figure of the primary urine derived cell culture to the 4th generation；
Fig. 3 is that the urine derived cell strain GE-HUC immortalized cultivates the cellular morphology figure to the 35th generation；
Fig. 4 is that the urine derived cell strain LV-HUC immortalized cultivates the cellular morphology figure to the 35th generation；
Fig. 5 is that the urine derived cell strain RV-HUC immortalized cultivates the cellular morphology figure to the 35th generation；
Fig. 6 is MTT comparative result figures；
Fig. 7 is the urine derived cell strain normal karyotype figure immortalized；
Fig. 8 is the urine derived cell strain LV-HUC chromosome abnormalities figures immortalized；
Fig. 9 is the urine derived cell strain RV-HUC chromosome abnormalities figures immortalized；
Figure 10 is primary urine cell beta galactosidase colored graph；
Figure 11 is the urine derived cell strain GE-HUC beta galactosidase colored graphs immortalized；
Figure 12 is the urine derived cell strain LV-HUC beta galactosidase colored graphs immortalized；
Figure 13 is urine derived cell strain RV-HUC β -- the galactosidase colored graph immortalized；
Specific embodiment
With reference to embodiment, the present invention is described further, but not limited to this.
Specific gene accession number and product related information are as follows in the present invention：
1 gene accession number of table
Product related information used in table 2
| Title | Brand | Article No. |
| P53 mutant | Prospec | PRO-301 |
The collection culture of embodiment 1, primary urine derived cell
The urine of 10ml-2L is collected, supernatant is abandoned in centrifugation (1010g, 5min), and precipitation is resuspended with containing dual anti-PBS
And carry out centrifugation again and abandon supernatant, Tissue Culture Plate (24 orifice plate) then is precipitated to using the resuspension of urine derived cell culture medium,
And 100 μ g/ml primocin are added in, 37 DEG C are then placed into, 5%CO2It is cultivated in cell incubator, during which not to it
Other operations are carried out, carry out changing liquid after 5 days, treat cell clone length to a certain size progress had digestive transfer culture.Cultivate the original being collected into
For urine derived cell form as shown in Figure 1, A is observed on the 5th day under the microscope for the collection culture of urine derived cell in Fig. 1
Adhered state, B is that urine derived cell collects culture to the cellular morphology of the 12nd day in Fig. 1, it can be seen that cell is in rice
Granular form.
(hyclone is with dimethyl sulfoxide (DMSO) by 9 for the primary urine cell frozen stock solution being collected into:1 prepares) cell is carried out
It freezes, freeze-stored cell in -196 DEG C of liquid nitrogen is positioned over after gradient cooling.It is synchronous to carry out STR partings and HLA Classification Identifications.
Embodiment 2, the structure of the urine derived cell strain immortalized
By the method that foreign gene or DNA fragmentation import cell and are integrated into cellular genome include the use of gene editing,
The method of slow virus carrier, retroviral vector etc..
1) gene editing method
1.1) special target sgRNA is found out
sgRNA-AAVS1-1：5’TATAAGGTGGTCCCAGCTCG 3’(SEQ ID NO：1)；
sgRNA-AAVS1-2：5’AGGGCCGGTTAATGTGGCTC 3’(SEQ ID NO：2).
1.2) sgRNA is building up to vector plasmid gRNA Cloning Vector
1.2.1) carrier preparation：AflII digestion gRNA Cloning Vector (Plasmid 41824, Adegene), then
Protruding terminus, last carrier end dephosphorylation are eliminated using mung-bean nuclease (#M0250S, NEB).
1.2.2) design and prepare the annealing segment of sgRNA
sgRNA-AAVS1-1：5’TATAAGGTGGTCCCAGCTCG 3’(SEQ ID NO：1)；
①.Clone-sgRNA-AAVS1-1-a：5’TTGTGGAAAGGACGAAACACCGTATAAGGTG 3’(SEQ ID
NO：3)；
②.Clone-sgRNA-AAVS1-1-b：5’GTCCCAGCTCGGTTTTAGAGCTAGAAATAGCAA 3’(SEQ
ID NO：4)；
③.Clone-sgRNA-AAVS1-1-c：5’CGGTGTTTCGTCCTTTCCACAA3’(SEQ ID NO：5)；
④.Clone-sgRNA-AAVS1-1-d：5’TTGCTATTTCTAGCTCTAAAACCGAGCTGGGACCACCTTATA
3’(SEQ ID NO：6)；
sgRNA-AAVS1-2：5’AGGGCCGGTTAATGTGGCTC 3’(SEQ ID NO：2)；
⑤.Clone-sgRNA-AAVS1-2-a：5’TTGTGGAAAGGACGAAACACCGAGGGCCGGT 3’(SEQ ID
NO：7)；
⑥.Clone-sgRNA-AAVS1-2-b：5’TAATGTGGCTCGTTTTAGAGCTAGAAATAGCAA 3’(SEQ
ID NO：8)；
⑦.Clone-sgRNA-AAVS1-2-c：5’CGGTGTTTCGTCCTTTCCACAA3’(SEQ ID NO：9)；
⑧.Clone-sgRNA-AAVS1-2-d：5’TTGCTATTTCTAGCTCTAAAACGAGCCACATTAACCGGCCCT
3’(SEQ ID NO：10).
2. 1. 3. 4. and 5. 6. 7. 8. annealing respectively, then end phosphorates processing.
1.2.3 SV40LT recombinant vectors) are built
Carrier and annealed product are connected and are transformed into DH5a competent cells, is smoothened after incubation containing antibiotic
On tablet, be incubated overnight in incubator, picking monoclonal bacterial strain is bacterium colony PCR, the inoculation of the electrophoresis result positive is small carry after
Send sequencing, be sequenced it is correct after carry out carrying in plasmid, obtain SV40LT genetic fragments after purification, by the method for restructuring,
SV40LT genes are inserted into Donor plasmids (band puromycin resistance gene Puro (R)) inside, are recycled after being handled with NruI digestions
SV40LT-Puro Donor segments.
1.3) SV40LT-Puro Donor are transformed into cell interior
1.3.1 primary urine derived cell) is recovered into 10cm wares, is carried out when cell culture to degree of converging 90% or so
Electricity turns.
1.3.2) primary 0.25% pancreatin of urine derived cell is digested, serum-containing media is terminated, and cell is whole
3,000,000 cells is taken to move to 15ml centrifuge tubes after only, supernatant is abandoned in centrifugation.
1.3.3 82 μ lBasic Nucleofector Solution For Mammalian) are sequentially added
Then Epithelial Cells and 18 μ l supplement 1 gently mixings add in plasmid Cas9D10A (5 μ g), sgRNA
Clone AAVS1 (3.5 μ g), SV40LT-puro (3.5 μ g).Afterwards again with rifle in 200 μ l by whole system mixing and transfering system
To electric revolving cup, electric revolving cup is transferred to electroporation T-020 programs electricity and is turned.
1.3.4 the cell after) electricity is turned is transferred in 6 orifice plates, is cultivated with the primary urine cell culture mediums of 3ml, is turned
Cell is positioned over 37 DEG C after shifting, 5%CO2It is cultivated in incubator.
1.4) cell purification
Urine derived cell after electricity is turned is changed to the urine cell containing 1.5 μ g/ml puromycin (puromycin)
Culture medium carries out screening and culturing, and not being transferred to the urine derived cell of SV40LT Donor segments can gradually die, and successfully be transferred to
The urine derived cell of SV40LT Donor segments can continue to increase in the presence of puromycin, be successfully established
The urine derived cell strain of immortalization, and this cell line is named as GE-HUC.
2) slow virus carrier method
2.1) structure of SV40LT gene eukaryotic expression vectors
By round pcr using 293T/17 cellular genomes as template, SV40LT genetic fragments are obtained, with genetic recombination skill
The method of art recombinates SV40LT genetic fragments and pSin-EF2 carriers, builds pSin-EF2-SV40LT plasmids, sequencing mirror
Determine recombinant plasmid.
2.2) cell transfecting
2.2.1 293T/17 cells) are inoculated with into two holes of 1 piece of six orifice plate, treat that adherence rate is left up to 70% afterwards for 24 hours
It is transfected when right.
2.2.2 following rotaring redyeing system) is configured.
Each component is added according to above-mentioned system, finally plus after 2xHBS, is quickly blown with pipettor 30 times, it is every after standing 2min
500 μ l of hole add in 293T/17 to be transfected, and each hole first renews fresh MEF culture mediums 1.5ml before transfection.
2.2.3 liquid is changed after) transfecting 6h, adds in 37 DEG C of fresh culture postposition, 5%CO2Continue to cultivate in incubator.
2.3) cell infection
2.3.1 urine derived cell) is inoculated with into 6cm wares, is felt afterwards when adherence rate is up to 30% or so for 24 hours
Dye.
2.3.2) after transfection for 24 hours, 0.45 μm of membrane filtration of the medium supernatant containing virus is collected, takes 3.5ml first
Subinfection urine cell, by 1:1000 add in polybrane, and before infection, urine cell first changes 1.5ml urine cell culture mediums,
293T/17, which adds in fresh MEF culture mediums, to be continued to cultivate, and is changed fresh culture after urine cell infection 6h, is put 37 DEG C, 5%CO2Training
It supports and continues to cultivate in case.
2.3.3 after) transfecting 48h, collected again containing viral supernatant, the second subinfection urine cell after filtering, after 6h
Fresh culture is changed, puts 37 DEG C, 5%CO2Continue to cultivate in incubator.
2.4) cell purification
By urine cell of the metainfective urine derived cell of virus containing 1.5 μ g/ml puromycin (puromycin)
Culture medium carries out screening and culturing, and not being transferred to the urine derived cell of pSin-EF2-SV40LT plasmids can gradually die, and successfully turn
Having entered the urine derived cell of pSin-EF2-SV40LT plasmids can continue to increase in the presence of puromycin, success
The urine derived cell strain immortalized is established, and this cell line is named as LV-HUC.
3) retroviral vector method
3.1) structure of SV40LT gene eukaryotic expression vectors
By round pcr using 293T/17 cellular genomes as template, SV40LT genetic fragments are obtained, with genetic recombination skill
The method of art recombinates SV40LT genetic fragments and pBabe-puro carriers, builds pBabe-puro-SV40LT plasmids, surveys
Sequence identifies recombinant plasmid.
3.2) cell transfecting
3.2.1) it is inoculated with 293T/17 cells 1.2x106Into 10cm wares, 24 it is small when after transfected.
3.2.2 following rotaring redyeing system) is configured.
Each component is added according to above-mentioned system, 2xHBS is finally added dropwise, 293T/ to be transfected is added in after standing 20min
17, P100 first renews fresh MEF culture mediums 9ml before transfection.
3.2.3 liquid is changed after) transfecting 6h, adds in 37 DEG C of fresh culture postposition, 5%CO2Continue to cultivate in incubator.
3.3) cell infection
3.3.1 primary urine derived cell) is inoculated with into 6cm wares, is carried out afterwards when adherence rate is up to 30% or so for 24 hours
Infection.
3.3.2) after transfection for 24 hours, 0.45 μm of membrane filtration of the medium supernatant containing virus is collected, takes 3.5ml first
Subinfection urine cell, by 1:1000 add in polybrane, and before infection, urine cell first changes 1.5ml urine cell culture mediums,
293T/17, which adds in fresh MEF culture mediums, to be continued to cultivate, and is changed fresh culture after urine cell infection 6h, is put 37 DEG C, 5%CO2Training
It supports and continues to cultivate in case.
3.3.3 after) transfecting 48h, collected again containing viral supernatant, the second subinfection urine cell after filtering, after 6h
Fresh culture is changed, puts 37 DEG C, 5%CO2Continue to cultivate in incubator.
3.4) cell purification
The metainfective urine derived cell of virus is changed to the primary urine cell containing 1.5 μ g/ml puromycin to train
It supports base and carries out screening and culturing, not being transferred to the urine derived cell of pBabe-puro-SV40LT plasmids can gradually die, and successfully turn
Having entered the urine derived cell of pBabe-puro-SV40LT plasmids can continue to increase in the presence of puromycin, into
Work(establishes the urine derived cell strain immortalized, and this cell line is named as RV-HUC.
Change SV40LT genes into urine derived cell strain that Myc can equally be immortalized.
Embodiment 3, the Colony Culture of the urine derived cell strain immortalized
Urine derived cell strain GE-HUC, LV-HUC and RV-HUC of the immortalization built in above-described embodiment 2 are distinguished
Carry out single cell clone culture.
1) it will cultivate to the urine derived cell strain of the immortalization in the 4th generation and be digested with pancreatin to single, be terminated with culture medium
Supernatant is abandoned in centrifugation afterwards, then is resuspended and counted with culture medium, and cell gradient is diluted to 500/ml, and 0.2ml is taken to be suspended from 15ml cultures
Base with volley of rifle fire kind plate, adds in 150 μ l cell suspensions in 96 orifice plates per hole.
2) 2 it is small when after, treat cell attachment then micro- Microscopic observation, write down and the position in the hole of only 1 cell.
3) 24 it is small when after the micro- above-mentioned mark of Microscopic observation hole, confirm have and only 1 clone hole be unicellular gram
It is grand.
4) single cell clone is observed daily, changes liquid every other day, after cell quantity increase, then is gone to bigger culture dish and is continued
Culture, with the single cell clone of the urine derived cell strain for being immortalized.
Cultivation results：Under the culture of the urine cell culture medium containing puromycin, GE-HUC being immortalized urines are thin
The monoclonal 25 of born of the same parents' strain, the monoclonal 32 of LV-HUC being immortalized urine cell lines, RV-HUC being immortalized urines
The monoclonal of cell line 30.
Embodiment 4, the morphologic observation and verification of the urine derived cell strain immortalized
1) the urine derived cell strain to the immortalization in the 35th generation is cultivated in observation in form
By the urine derived cell strain of immortalization respectively in culture dish, with the urine cell culture medium containing puromycin
It is cultivated, replaces within every two days a subculture, cell grows to 80% left back, passes on, micro- Microscopic observation comparison culture to the
The form of the primary urine derived cell in 4 generations and the urine derived cell cultivated to the immortalization in the 35th generation.
Fig. 2 is cellular morphology of the primary urine derived cell culture to the 4th generation, it can clearly be seen that cell attachment state
Difference, aging, Fig. 3, Fig. 4, Fig. 5 are respectively that urine derived cell strain GE-HUC, LV-HUC, RV-HUC culture immortalized is arrived
The cellular morphology in the 35th generation, all smaller in the urine derived cell plant shape state of immortalization, adhered state is good, and still without substantially declining
Old phenomenon.
2) mtt assay detection cell proliferation rate
MTT testing principles are that the succinate dehydrogenase in living cells mitochondria can make exogenous MTT be reduced to water-insoluble
Bluish violet Jie Jing Jia Za and be deposited in cell, and dead cell is without this function.DMSO can dissolve Jia Za in cell, with enzyme mark
Instrument measures its absorbance value, and in the range of certain cell number, it is directly proportional to viable count that MTT crystallizes the amount to be formed.According to measuring
Absorbance (OD values) judge living cells quantity.
The urine derived cell to the primary urine derived cell and the immortalization of culture to the 35th generation in the 4th generation will be cultivated
GE-HUC, LV-HUC and RV-HUC press 2x103A cell per well is inoculated in 96 orifice plates, and per 150 μ l culture mediums of hole, every group 5 multiple
Hole, respectively culture for 24 hours, after 48h, 72h, 96h and 120h, per hole add in 15 μ l tetramethyl azo azoles salts (MTT, 5mg/ml),
Continue culture 4 it is small when after discard whole culture mediums, add in 150 μ l dimethyl sulfoxide (DMSO)s (DMSO), shake 10min in concussion instrument, use
Microplate reader surveys the OD values per hole, calculates and is compared per cell mean.
MTT results (Fig. 6) are shown：A groups are the 4th generation primary urine derived cell, and B, C, D group are respectively to immortalize in the 35th generation
Urine derived cell GE-HUC, LV-HUC, RV-HUC, the results show A group cell later stages increase apparent slack-off, and B, C, D group are carefully
Born of the same parents almost increase in logarithm, and 3 times of the OD values almost A groups to the 5th day B, C, D group.
3) caryogram is identified
Randomly select the single cell clone of the urine derived cell strain for the immortalization cultivated in embodiment 3, GE-HUC,
Each 7 plants of LV-HUC and RV-HUC, cultivates respectively to the 35th generation and does caryogram identification.
3.1) cell prepares
Growth conditions are good, and stand density is between 80~90%.
3.2) colchicine is handled
Culture terminates the colchicine that the preceding amount in culture medium adds in final concentration of 0.2 μ g/mL, is handled in 37 DEG C of incubators
100~130min.
3.3) Hypotonic treatment
It after colchicine has been handled, first inhales and abandons culture solution, washed twice with PBS, add in the digestion of 0.25% pancreatin, gently beat
Culture dish makes the cell detachment not fallen off, and adds in MEF and terminates digestion, is transferred to 15mL centrifuge tubes with suction pipe absorption, centrifuges
(1200rpm, 5min) collects cell, then adds in the 0.075mol/L KCL solution 7mL of 37 DEG C of preheatings, is blown and beaten with suction pipe into thin
Born of the same parents' suspension puts 37 DEG C of water bath processing 18-28min.
3.4) pre-fix and fix
(methanol presses 3 to Fresh Kano fixer with glacial acetic acid:1 prepare), with rubber head dropper add in about 1mL fixers into
Row pre-fixes 10min.After pre-fixing, supernatant is abandoned in centrifugation (1200rpm, 5min), is added in the fresh fixers of about 7mL, is used rubber head
Dropper is gently beaten, and 40min is fixed in 37 DEG C of water-baths.
3.5) piece is dripped
After the completion of fixation, most of fixer is abandoned with the suction of rubber head dropper after centrifugation (1200rpm, 5min), part is stayed to fix
Cell is resuspended in liquid (determining how much stay liquid according to the amount of cell), is dripped freezing 30cm or so distances above clean glass slide
Piece.
3.6) piece is baked
Glass slide is moved into 75 DEG C of baking ovens baking 3h at once after dripping piece.
3.7) (G shows band) is dyed
0.03g pancreas enzyme powders are added in toward 55mL physiological saline, are gently shaken up, it is about 7.2 to adjust its pH with 3%Tris.It will system
Piece be put into pancreatin digestive juice handle 8 seconds after, be put into physiological saline rapidly and terminate its digestion, place into Giemsa dye liquors dyeing 5~
Then 10min presss from both sides out slide with tweezers, two sides, drying at room temperature or hair dryer drying are gently rinsed with tap water.
3.8) microscopy analysis
It after slide is done, checks under the microscope, first finds good split coil method with low power lens, then seen with high power oil mirror
It examines, chromosome number, banding pattern is analyzed, each cell is divided into 20 division visuals field of analysis.
Analysis result：The urine derived cell strain GE-HUC caryogram of immortalization is normal, the urine derived cell of immortalization
There are 2 exceptions (abnormal rate 28.6%) in the results of karyotype of strain LV-HUC, the urine derived cell strain RV-HUC's of immortalization
There is 1 exception (abnormal rate 14.3%) in results of karyotype.Fig. 7 is the normal karyotype result of the urine derived cell strain immortalized
Figure, Fig. 8 are the abnormal karyotype result figure of the urine derived cell strain LV-HUC immortalized, and Fig. 9 is that the urine source immortalized is thin
The abnormal karyotype result figure of born of the same parents RV-HUC.
4) oncogenicity detects
Take the logarithm growth period culture to the primary urine derived cell and A-498 cells in the 4th generation, then randomly select culture
Each 7 plants of urine derived cell GE-HUC, LV-HUC, RV-HUC immortalized into the embodiment 3 in the 35th generation is used after pancreatin digestion
Serum-free DMEM is prepared into single cell suspension.10 are taken respectively7The single cell suspension of/ml is injected into the flesh of NOD-SCID mouse four limbs
Meat is subcutaneous, then carries out observation culture.Mouse can be observed after one month into knurl or death condition such as following table：
Table 3 detects for the oncogenicity of mouse
The oncogenicity testing result of table 3 shows the urine source of the immortalization obtained by retroviral vector method
Cell (RV-HUC) passes through the urine derived cell (LV- of the obtained immortalization of slow virus carrier method there are oncogenicity risk
HUC oncogenicity risk) is not embodied in the detection experiment of this oncogenicity, but can be caused death to mouse, and passes through gene editing
It the no oncogenicity of urine derived cell (GE-HUC) for the immortalization that method obtains and will not cause death to mouse, show to pass through
The urine derived cell for the immortalization that gene editing method obtains is compared with passing through slow virus carrier method or retroviral vector side
The cell that method obtains can more maintain the characteristic of primary urine cell.
5) betagalactosidase activity
Urine derived cell GE-HUC, LV- of the 4th generation primary urine derived cell and the 35th generation immortalization are digested respectively
HUC and RV-HUC is simultaneously counted, per hole (24 orifice plate) bed board 2x103A cell is cultivated using urine cell culture medium,
Day7 carries out aging detection.When carrying out aging detection, first add in PBS and washed, washed again with PBS after fixed, so
The sweet enzyme dyeing liquid of beta galactose of Fresh is added in afterwards, and 37 DEG C of overnight incubations are dyed, second day micro- Microscopic observation dye
Pornographic condition.Figure 10 be primary urine cell dyeing figure, Figure 11,12,13 be respectively immortalize urine derived cell GE-HUC,
The colored graph of LV-HUC and RV-HUC.It can observe to obtain primary urine derived cell by figure and just decline in more early algebraically
Always, it is still more young after more generation is cultivated to immortalize urine derived cell.
6) recovery Efficiency testing
Urine derived cell GE-HUC, LV-HUC and RV-HUC of immortalization is in 37 DEG C, 5%CO2In incubator, with containing
The urine cell culture medium of puromycin carries out cellar culture, 0.25% pancreatin had digestive transfer culture of cell, with frozen stock solution (tire ox
Serum presses 9 with dimethyl sulfoxide (DMSO):1 prepares) cell is frozen, it is positioned in -196 DEG C of liquid nitrogen and freezes after gradient cooling
Cell.Carried out recovery culture after 1 month, adherent rate is all 95% or so after cell recovery, growth that can be steady in a long-term
And passage.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>White LLai Biology Pharmacy Co., Ltd, Wang Linli
<120>A kind of urine derived cell strain of immortalization and its construction method
<130>
<160> 10
<170> PatentIn version 3.5
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Claims (7)
1. a kind of urine derived cell strain of immortalization, it is characterised in that：The cell line derives from urine specimen, possesses external
Infinite multiplication or the ability close to infinite multiplication.
2. the urine derived cell strain of immortalization according to claim 1, it is characterised in that：The urine of the immortalization is come
Source cell strain is that importing external source immutalizing gene or DNA segment are built-up in urine derived cell, the immutalizing gene
Or DNA segment includes at least one of SV40LT, Bmi1, E6, E7, p53 mutant, Myc, c-Jun and Mdm2.
3. the construction method of the urine derived cell strain of a kind of immortalization, which is characterized in that comprise the following steps：It will carry forever
The Transfected Recombinant Plasmid of biochemical gene or DNA fragmentation is into the urine derived cell strain that immortalization is built into urine derived cell.
4. construction method according to claim 3, it is characterised in that：The immutalizing gene or DNA fragmentation include
At least one of SV40LT, Bmi1, E6, E7, p53 mutant, Myc, c-Jun and Mdm2.
5. according to claim 3-4 any one of them construction methods, it is characterised in that：Immutalizing gene or DNA will be carried
The method of the Transfected Recombinant Plasmid of segment into urine derived cell includes the use of retroviral vector, slow virus carrier, gene
At least one of editor.
6. construction method according to claim 5, it is characterised in that：The weight of immutalizing gene or DNA fragmentation will be carried
Group plasmid transfection is into urine derived cell and to be integrated into the method for cellular genome be preferably gene editing.
7. the urine derived cell strains of any one of the claim 1-2 immortalizations is built in the urine derived cell strain of immortalization
Application in storehouse.
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| PCT/CN2018/101084 WO2019140900A1 (en) | 2018-01-19 | 2018-08-17 | Immortalized urine-source cell strain and building method thereof |
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| WO2019140900A1 (en) * | 2018-01-19 | 2019-07-25 | 皓昇莱生物制药有限公司 | Immortalized urine-source cell strain and building method thereof |
| CN112941034A (en) * | 2021-04-08 | 2021-06-11 | 江苏易诺维生物医学研究院有限公司 | Construction method of immortalized human umbilical cord mesenchymal stem cell line |
| CN114134118A (en) * | 2021-11-22 | 2022-03-04 | 南方医科大学南方医院 | Immortalized human laryngeal ring posterior region cell and construction method thereof |
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| CN101617043A (en) * | 2007-10-31 | 2009-12-30 | 国立大学法人京都大学 | Nuclear reprogramming method |
| CN105483088A (en) * | 2005-10-18 | 2016-04-13 | 国家犹太健康中心 | Conditionally immortalized long-term stem cells and methods of making and using such cells |
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| EP2832851B1 (en) * | 2012-03-28 | 2019-04-17 | Quarrymen & Co. Inc. | Immortalized stem cells and medicinal composition and medicinal preparation comprising product thereof as active ingredient |
| CN108070567A (en) * | 2018-01-19 | 2018-05-25 | 皓昇莱生物制药有限公司 | A kind of urine derived cell strain of immortalization and its construction method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483088A (en) * | 2005-10-18 | 2016-04-13 | 国家犹太健康中心 | Conditionally immortalized long-term stem cells and methods of making and using such cells |
| CN101617043A (en) * | 2007-10-31 | 2009-12-30 | 国立大学法人京都大学 | Nuclear reprogramming method |
Non-Patent Citations (2)
| Title |
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| MARTIJN J. WILMER: "Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters", 《CELII AND TISSUE RESEARCH》 * |
| 温晟: "SV40T抗原基因介导尿源性干细胞永生化的建立", 《中华小儿外科杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019140900A1 (en) * | 2018-01-19 | 2019-07-25 | 皓昇莱生物制药有限公司 | Immortalized urine-source cell strain and building method thereof |
| CN112941034A (en) * | 2021-04-08 | 2021-06-11 | 江苏易诺维生物医学研究院有限公司 | Construction method of immortalized human umbilical cord mesenchymal stem cell line |
| CN114134118A (en) * | 2021-11-22 | 2022-03-04 | 南方医科大学南方医院 | Immortalized human laryngeal ring posterior region cell and construction method thereof |
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