CN114085874B - Method for preparing immortalized liver cells with reversible liver functions and application thereof - Google Patents

Abstract

The invention discloses a method for preparing immortalized liver cells with reversible liver functions and application thereof, relating to the field of biomedicine. The invention applies the Tet-on system to the liver cells, and the functions of the liver cells are strengthened by the Tet-on system. The Tet-on system has the characteristics of high efficiency, accuracy, safety and reversibility. Therefore, the novel immortalized liver cell is prepared, and the reversible regulation of the liver cell function can be realized by adding or removing tetracycline analogues, so that the reversible enhancement of the liver cell function is promoted. The preparation method is simple and easy to implement, the culture cost is low, and the seed cells are derived from immortalized liver cells and are easy to clinically transform.

Description

Method for preparing immortalized liver cells with reversible liver functions and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a method for preparing immortalized liver cells with reversible liver functions and application thereof.

Background

The appropriate liver cell line is becoming more and more important in bioartificial liver technology, pharmacological toxicology research and liver tissue engineering research. An ideal hepatocyte source should have intact liver function, good proliferative capacity and no tumorigenic, infection risk. The conventional hepatocyte sources comprise primary human hepatocytes, primary pig hepatocytes, human hepatoma cell lines HepG2, HepaRG, immortalized cells, stem cell differentiated hepatocytes and the like. Although these kinds of hepatocytes have a certain liver function in vitro, they have application obstacles such as insufficient maturity, limited expansion, unstable function, xenograft rejection, low biosafety, and tumorigenic risk, respectively.

Chinese patent application publication nos. CN110438157B and CN105521482A disclose methods for further inducing differentiation of hepatocytes by using different hepatocyte-specific transcription factors alone or in combination, but these methods or seed cells are derived from liver cancer cells, or the used culture system is expensive, and clinical transformation is difficult.

Chinese patent application publication nos. CN105925609B and CN107557389A disclose methods for regulating differentiation of stem cells into different somatic cells by controllable induction of overexpression of different target foreign genes. The former promotes differentiation of pluripotent stem cells into hematopoietic cells, and the latter promotes differentiation of mesenchymal stem cells into osteoblasts.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The present invention aims to provide a method for preparing immortalized hepatocytes whose liver functions are reversible and applications thereof to solve the above-mentioned technical problems.

The inventors applied the Tet-on system to hepatocytes, and enhanced the function of hepatocytes by improving the Tet-on system. Therefore, a novel immortalized liver cell is prepared, and the reversible regulation of the liver cell function can be realized by adding or removing tetracycline analogues, so that the enhancement of the reversible function of the liver cell is promoted.

The invention is realized by the following steps:

the invention provides a method for preparing immortalized liver cells with reversible liver functions, which comprises the steps of infecting human immortalized liver cells with packaged lentiviruses containing a Tet-on system for regulating and controlling the transcription of the liver cells, and then carrying out resistance screening and amplification subculture on the cells obtained after virus transfection by using a screening agent and an amplification culture medium to obtain a reversible immortalized liver cell line with strengthened liver functions; the immortalized liver cell line is in a liver function strengthening state by adding the tetracycline analogue, and is in a normal liver cell proliferation state by removing the tetracycline analogue;

the Tet-on system for regulating and controlling the transcription of the hepatocyte comprises a regulation expression box and a reaction expression box, wherein the regulation expression box comprises a liver specific promoter and an antisense tetracycline activator, and the reaction expression box comprises a tetracycline inducible promoter and a transcription factor coding gene; transcription factor encoding genes include, but are not limited to, the FOXA3 gene, the CEBPA gene, and the GATA6 gene.

The preparation method of the immortalized liver cell with reversible liver function provided by the invention has the characteristics of high efficiency, accuracy, safety and reversibility.

The high efficiency is reflected in that: the expression level of the target gene is lower in the absence of induction, and the target gene is expressed in the presence of tetracycline analogue induction. The target gene includes a transcription factor coding gene and a screening gene. The concrete expression is as follows: in the absence of a tetracycline analogue, the antisense tetracycline activator fails to bind to the TRE. Since the reaction expression cassette lacks an enhancer, when a reverse tetracycline activator (rtTA) is not bound to a TRE, the tetracycline-inducible promoter cannot promote the expression of the target gene, and thus it appears that the gene expression is inhibited. In the presence of tetracycline analogs, the antisense tetracycline activator is capable of binding to the TRE, thereby activating the tetracycline-inducible promoter and allowing expression of the gene of interest.

The precise regulation is realized by reversible regulation of liver cell function enhancement through controlling dosage and administration time of tetracycline analogue.

The safety is realized by adopting a tetracycline analogue with lower dose to obtain good effect, and Tet-on is applied for years and is safe and reliable.

The induction can be initiated for multiple times by repeated addition of induction.

The inventor simultaneously targets and over-expresses three hepatocyte-related transcription factors FOXA3, CEBPA and GATA6 by inducing a promoter through a tetracycline analogue, so that the immortalized hepatic cells realize reversible hepatic function enhancement. It should be noted that the above transcription factors were obtained by the inventors through a large and long-term screening, and the inventors found that the hepatic function enhancement of the immortalized hepatocyte-like cells can be achieved only by simultaneously overexpressing the above three hepatocyte-associated transcription factors.

In a preferred embodiment of the present invention, the human immortalized liver cells are derived from a human immortalized liver cell line as a basal seed cell; performing a culture preparation of the basal seed cells prior to the lentivirus infection, the culture preparation of the basal seed cells comprising: culturing the human immortalized liver cells to obtain the basal seed cells with the confluence rate of 50-70%. The confluence rate is the percentage of the area occupied by the cells to the culture surface area after the cells are attached and fully expanded.

The tetracycline analogue is chlortetracycline, oxytetracycline, minocycline, doxycycline or doxycycline.

In a preferred embodiment of the present invention, the ratio of the packaged lentivirus to the human immortalized liver cells is: every 5X 10⁵Number of human immortalized hepatocytes and 1X 10⁷-2.5×10⁷A number of packaged lentivirus solutions were mixed.

Alternatively, the human immortalized liver cell line used as the basal seed cell may be C3A.

The inventors found that when tetracycline analogue was present, the cells were in a hepatic-enhanced state, the microscopic cell morphology was typical hepatocyte morphology (polygonal), grown uniformly flat, expressed normal hepatocyte markers, and increased the synthesis capacity of ALB (albumin), AAT (α 1-antitrypsin), urea, and coagulation factor 7; when doxycycline is removed, cells are in a normal state, the microscopic form is epithelial cell-like, and the cells grow in an island-like aggregation manner, so that the synthetic ability of AAT, urea and blood coagulation factor 7 is not excellent. The function of the immortalized liver cells with reversible liver function prepared by the preparation method provided by the invention is strengthened.

In one embodiment, the tetracycline analogue is chlortetracycline, oxytetracycline, minocycline, doxycycline or doxycycline.

In a preferred embodiment of the application of the invention, after 48-72 hours after the immortalized human hepatocytes are infected, the immortalized human hepatocytes are subcultured by using a medium containing a screening agent; the generation number of subculture is 3-4.

In a preferred embodiment of the present invention, the screening agent is puromycin (puro), phleomycin (phleomycin), blasticidin (blastcidin), hygromycin (hygb) or neomycin (neo).

The screening agent can further screen cells successfully infected by the virus, and is favorable for creating good culture conditions for the subsequent establishment of the immortalized cell line.

In a preferred embodiment of the present invention, the regulatory expression cassette further comprises a first selection gene, a post-transcriptional regulatory element and an adapter element, wherein the liver-specific promoter, the antisense tetracycline activator, the adapter element, the first selection gene and the post-transcriptional regulatory element are connected in series in the 5 '-3' direction;

preferably, the liver-specific promoter is selected from the group consisting of CMV promoter, PGK promoter, albumin promoter, apolipoprotein E promoter, phosphoenolpyruvate carboxykinase promoter, alpha-I-antitrypsin promoter, thyroid hormone binding globulin promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, IGF-II promoter, factor VIII promoter, HBV basic core protein promoter, HBV pre-s 2 protein promoter, thyroxine-binding globulin promoter, hybrid promoter of HCR-Ap0CII, HCR-hAAT hybrid promoter, AAT promoter in combination with enhancer elements of mouse albumin gene, low density lipoprotein promoter, pyruvate kinase promoter, lecithin-cholesterol acyltransferase promoter, apolipoprotein H promoter, iron transfer protein promoter, alpha-fetoprotein promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, IGF-II promoter, factor VIII promoter, HBV basic core protein promoter, HBV pre-s 2 protein promoter, thyroxine-binding globulin promoter, hybrid promoter of HCR-Ap0CII, HCR-hAAT promoter, AAT promoter in combination with enhancer elements of mouse albumin gene, low density lipoprotein promoter, pyruvate kinase promoter, lecithin-cholesterol acyltransferase promoter, apolipoprotein H promoter, iron transfer protein promoter, and alpha-fetoprotein-alpha-, Any one of a transthyretin promoter, promoters of alpha-fibrinogen and beta-fibrinogen, an alpha-I-antichymotrypsin promoter, an alpha-2-HS glycoprotein promoter, a haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a complement protein promoter, a promoter of a complement C3 activator, a hemopexin promoter and an alpha-I-acidic glycoprotein promoter;

the first selection gene is selected from puromycin resistance gene, phleomycin resistance gene, blasticidin resistance gene, hygromycin resistance gene or neomycin resistance gene.

In a preferred embodiment of the present invention, the tetracycline-inducible promoter and the transcription factor-encoding gene are sequentially connected in series along the 5 '-3' direction, and the reaction expression cassette further comprises a second selection gene located downstream of the transcription factor-encoding gene;

the second selection gene is selected from eukaryotic resistance genes;

preferably, the eukaryotic resistance gene is selected from the group consisting of puromycin resistance gene, phleomycin resistance gene, blasticidin resistance gene, hygromycin resistance gene or neomycin resistance gene. Antibiotic resistance genes (encoding antibiotic resistance proteins) provide resistance to specific antibiotics such that only cells expressing these resistance genes can survive and multiply.

In a preferred embodiment of the present invention, the tetracycline-inducible promoter is selected from the TRE3G promoter.

The invention also provides application of the immortalized liver cells with reversible liver functions prepared by the method in the field of bioartificial liver reactors. The immortalized liver cell with reversible liver function prepared by the method can be used as a therapeutic liver cell to be applied to a bioartificial liver reactor.

The invention also provides an application of the Tet-on system for regulating the transcription of the liver cells in the preparation of immortalized liver cells with reversible liver functions, wherein the Tet-on system for regulating the transcription of the liver cells comprises a regulating expression box and a reaction expression box, the regulating expression box comprises a liver specific promoter and an antisense tetracycline activator, and the reaction expression box comprises a tetracycline inducible promoter and a transcription factor coding gene; transcription factor encoding genes include the FOXA3 gene, the CEBPA gene and the GATA6 gene.

The invention has the following beneficial effects:

the invention applies the Tet-on system to the liver cells, and enhances the functions of the liver cells by improving the Tet-on system. The Tet-on system has the characteristics of high efficiency, accuracy, safety and reversibility. Therefore, a novel immortalized liver cell is prepared, and the reversible regulation of the liver cell function can be realized by adding or removing tetracycline analogues, so that the enhancement of the reversible function of the liver cell is promoted. The preparation method is simple and easy to implement, the culture cost is low, and the seed cells are derived from immortalized cells and are easy to clinically transform.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a diagram showing the morphology and growth of four groups of cells under a microscope;

FIG. 2 is a statistical chart of ELISA detection results of ALB in four groups of cell culture supernatants;

FIG. 3 is a statistical chart of ELISA detection results of AAT in four groups of cell culture supernatants;

FIG. 4 is a statistical chart of ELISA detection results of urea in four groups of cell culture supernatants;

FIG. 5 is a statistical chart of the ELISA detection results of coagulation factor 7 in the four groups of cell culture supernatants;

FIG. 6 is a vector diagram of a regulatory expression cassette;

FIG. 7 is a vector diagram of the reaction expression cassette.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a method of preparing immortalized hepatocytes whose liver function is reversible. The Tet-on system for regulating and controlling the transcription of the liver cells comprises a regulating expression box and a reaction expression box, wherein the regulating expression box comprises a liver specific promoter and an antisense tetracycline activator, and the reaction expression box comprises a tetracycline inducible promoter and a liver cell transcription factor coding gene; hepatocyte transcription factor encoding genes include the FOXA3 gene, the CEBPA gene and the GATA6 gene. The vector diagram of the regulatory expression cassette is shown in FIG. 6, the sequence is shown in SEQ ID NO.1, the vector diagram of the response expression cassette is shown in FIG. 7, and the sequence is shown in SEQ ID NO. 2. The lentivirus sequences are delivered to Yunzhou Biotechnology (Guangzhou) GmbH for virus packaging.

The method for preparing immortalized liver cells with reversible liver functions comprises the following steps:

(1) preparing basal seed cells: well-conditioned cells of interest (C3A, from ATCC cell bank) were plated onto 12-well plates, typically with a cell confluence of between 50% and 70% at the next day of viral infection.

(2) Infection of basal seed cells: before infection, the virus was removed from the freezer and thawed, the original medium was aspirated off the cells and replaced with half the amount of fresh medium per 5X 10⁵The number of human immortalized hepatocytes added was 2.5X 10⁷The slow virus liquid in the amount is mixed evenly in a gentle 8-character mode. The next day after infection (about 12 hours), the virus-containing culture medium was aspirated off and replaced with fresh complete culture mediumThe culture was continued at 37 ℃.

(3) Screening target cells: at 48 hours post-infection, the stably transduced cell lines were selected by replacing fresh complete medium containing Puromycin (Puromycin) at the appropriate concentration (working concentration 1-10 ug/mL). Complete cultures containing puromycin were changed every 2-3 days until unsuccessfully transfected cells were killed by puromycin. The infected and selected cells were then passaged and maintained in selection culture by continued application of puromycin. After continuous screening and 3 generations, the stable cell strain is frozen and preserved.

(4) Observation of the cells of interest: successfully transfected cells of interest and untransfected basal seed cells were cultured for 7 days using medium with or without doxycycline (purchased from Shanghai Binyan Biotechnology Ltd.). After 7 days, the morphology and growth of the cells were observed under a microscope.

Experimental example 1

Microscopic field pattern referring to fig. 1, the successfully transfected target cells in fig. 1 were in a normal state after being cultured in doxycycline-free medium for 7 days, and the microscopic morphology was epithelial-like and grown in island-like aggregates, and labeled as C3A-FOXA3/CEBPA/GATA 6-CTRL. And after the target cells which are successfully transfected are cultured in a Doxycycline (DOX) containing culture medium for 7 days, the microscopic cell morphology is the typical hepatocyte morphology (polygon), and the cells grow evenly and flatly and are marked as C3A-FOXA3/CEBPA/GATA6-DOX1000ng-day 7.

And after the target cells which are not successfully transfected are cultured in a doxycycline-free culture medium for 7 days, the cells are in a normal state, and have an epithelial cell-like shape under the microscope, grow in an island-shaped aggregation manner and are marked as C3A-CTRL. And after the target cells which are not successfully transfected are cultured in a doxycycline-containing culture medium for 7 days, the shape of the under-mirror cells is maintained and is in a normal state, and the under-mirror cells are epithelial cell-like and grow in an island-shaped aggregation manner and are marked as C3A-DOX 1000ng-day 7.

The results indicate that morphological changes in transfected successful cells are a result of the opening of the tet-on system, independent of doxycycline stimulation.

Experimental example 2

In this example, four groups of cells were tested for the ability to synthesize Albumin (ALB), alpha 1-antitrypsin (AAT), UREA (UREA) and coagulation factor 7 (FVII) by ELISA after 7 days of culture, and the number of cells, the volume of culture supernatant and the culture time were unchanged during the experiment, and the single variable was the cell).

The results showed that when the target cells successfully transfected were in a state of liver enhancement (doxycycline addition), normal hepatocyte markers were expressed, and the synthesis ability of ALB (see fig. 2), AAT (see fig. 3), urea (see fig. 4) and coagulation factor 7 (see fig. 5) was improved. When doxycycline is removed, the synthesis capacity of cellular AAT, urea, and coagulation factor 7 is not excellent.

The experimental example shows that the immortalized liver cells with reversible liver functions prepared by the Tet-on system provided by the invention have the effect of promoting the enhancement of the hepatic function, and the expression of three hepatocyte-related transcription factors FOXA3/CEBPA/GATA6 can be controlled in a targeted manner by controlling tetracycline analogues, so that the reversible function enhancement process of the immortalized liver cells is controlled. So that the immortalized hepatic cell can be switched between the normal state and the hepatic-to-function strengthening state.

Aiming at a target cell line C3A, the best effect of the virus tet-on system provided by the invention is that the DOX concentration is 500-1000 ng, and the adding culture time is 5-7 days.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Guangdong Qianhui Biotech Co., Ltd

<120> method for preparing immortalized liver cells with reversible liver function and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 9200

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agccgcgagt gcgtgaactg cggctccatc cagacgccgc tgtggcggcg ggacggcacc 5940

ggccactacc tgtgcaacgc ctgcgggctc tacagcaaga tgaacggcct cagccggccc 6000

ctcatcaagc cgcagaagcg cgtgccttca tcacggcggc ttggattgtc ctgtgccaac 6060

tgtcacacca caactaccac cttatggcgc agaaacgccg agggtgaacc cgtgtgcaat 6120

gcttgtggac tctacatgaa actccatggg gtgcccagac cacttgctat gaaaaaagag 6180

ggaattcaaa ccaggaaacg aaaacctaag aacataaata aatcaaagac ttgctctggt 6240

aatagcaata attccattcc catgactcca acttccacct cttctaactc agatgattgc 6300

agcaaaaata cttcccccac aacacaacct acagcctcag gggcgggtgc cccggtgatg 6360

actggtgcgg gagagagcac caatcccgag aacagcgagc tcaagtattc gggtcaagat 6420

gggctctaca taggcgtcag tctcgcctcg ccggccgaag tcacgtcctc cgtgcgaccg 6480

gattcctggt gcgccctggc cctggcctga acccagcttt cttgtacaaa gtggtgataa 6540

tcgaattccg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 6600

aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 6660

attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 6720

tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 6780

gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 6840

ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 6900

ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt 6960

ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 7020

ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 7080

cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 7140

catcgggaat tcccgcggtt cgaattctac cgggtagggg aggcgctttt cccaaggcag 7200

tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg 7260

cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc 7320

ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg 7380

tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc 7440

accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc 7500

ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg 7560

ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa 7620

aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctcacgt 7680

ggccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag 7740

ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga 7800

tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg 7860

gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac 7920

gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt 7980

gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc 8040

caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg 8100

tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc 8160

cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt 8220

caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg 8280

tgcctgaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt 8340

taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt 8400

ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 8460

tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg 8520

cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga 8580

aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga 8640

aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa 8700

gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 8760

tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc 8820

gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 8880

tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg 8940

aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac 9000

gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 9060

cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 9120

accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc 9180

ggcgcattaa gcgcggcggg 9200

<210> 2

<211> 8608

<212> DNA

<213> Artificial sequence

<400> 2

aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60

tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120

tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180

gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240

gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300

tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360

taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420

aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480

gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540

actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600

attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660

aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720

gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780

tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840

atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900

aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960

caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020

acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080

tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140

gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200

ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260

ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320

ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380

atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta 1800

gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 1860

atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1920

actagtgatt atcggatcaa ctttgtatag aaaagttgta gttattaata gtaatcaatt 1980

acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 2040

ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 2100

cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 2160

actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 2220

aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 2280

acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 2340

tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 2400

gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 2460

aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 2520

agagctggtt tagtgaaccg tcagatccaa gtttgtacaa aaaagcaggc tgccaccatg 2580

tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa tggagtcggt 2640

atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg 2700

tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac 2760

aggcatcata cccactcctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac 2820

aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc 2880

ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt 2940

cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca 3000

ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct 3060

accaccgatt ctatgccccc acttctgaaa caagcaattg agctgttcga ccggcaggga 3120

gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta 3180

aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca 3240

gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt 3300

gaccttgaca tgctccccgg gggaagcgga gagggcaggg gaagtcttct aacatgcggg 3360

gacgtggagg aaaatcccgg ccccatgaaa aagcctgaac tcaccgcgac gtctgtcgag 3420

aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa 3480

gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc 3540

tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc 3600

ccgattccgg aagtgcttga cattggggaa tttagcgaga gcctgaccta ttgcatctcc 3660

cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg 3720

cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg 3780

ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc 3840

gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg 3900

tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg 3960

cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca 4020

gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc 4080

ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg 4140

catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac 4200

caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga 4260

tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga 4320

agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc 4380

cccagcactc gtccgagggc aaaggaatag acccagcttt cttgtacaaa gtggtgataa 4440

tcgaattccg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 4500

aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 4560

attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 4620

tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 4680

gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 4740

ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 4800

ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt 4860

ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 4920

ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 4980

cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 5040

catcgggaat tcccgcggtt cgctttaaga ccaatgactt acaaggcagc tgtagatctt 5100

agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa 5160

gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc 5220

tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 5280

gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag 5340

tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa 5400

cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt 5460

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 5520

agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc 5580

ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 5640

gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 5700

agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga 5760

gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 5820

cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 5880

agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc 5940

gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 6000

acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 6060

cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 6120

tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 6180

gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 6240

cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 6300

gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 6360

gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc 6420

ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 6480

taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 6540

cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 6600

acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 6660

ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 6720

atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 6780

gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 6840

acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 6900

atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 6960

accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 7020

ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 7080

acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 7140

gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 7200

tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 7260

ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 7320

actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 7380

taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 7440

tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 7500

gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 7560

cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 7620

gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 7680

gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 7740

tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 7800

ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 7860

cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 7920

gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agagagaaag 7980

gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 8040

gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 8100

cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 8160

tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 8220

cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 8280

cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 8340

ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 8400

tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 8460

caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 8520

tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 8580

agggaacaaa agctggagct gcaagctt 8608

Claims (9)

1. A method for preparing immortalized liver cells with reversible liver functions is characterized in that packaged lentiviruses containing a Tet-on system for regulating and controlling the transcription of the liver cells infect the immortalized liver cells of a human, and then resistance screening and amplification subculture are carried out on the cells after virus transfection by utilizing a screening agent and an amplification culture medium to obtain an immortalized liver cell line with reversible liver functions; the immortalized liver cell line is in a liver function strengthening state by adding the tetracycline analogue, and is in a normal liver cell proliferation state by removing the tetracycline analogue;

the Tet-on system for regulating and controlling the transcription of the hepatocyte comprises a regulation expression box and a reaction expression box, wherein the regulation expression box comprises a liver specific promoter and an antisense tetracycline activator, and the reaction expression box comprises a tetracycline inducible promoter and a transcription factor coding gene; the transcription factor coding gene comprises FOXA3 gene, CEBPA gene and GATA6 gene, the regulation expression box also comprises a first screening gene, a post-transcriptional regulatory element and an engagement element, and the liver specificity promoter, the antisense tetracycline activator, the engagement element, the first screening gene and the post-transcriptional regulatory element are connected in series in sequence along the 5 '-3' direction;

the tetracycline-inducible promoter and the transcription factor coding gene are sequentially connected in series along the 5 '-3' direction, and the reaction expression cassette further comprises a second screening gene which is positioned at the downstream of the transcription factor coding gene;

the tetracycline analogue is chlortetracycline, oxytetracycline, minocycline, doxycycline or doxycycline.

2. The method of claim 1, wherein the human immortalized liver cells are cells derived from a human immortalized liver cell line as a basal seed cell; performing culture preparation of a basal seed cell prior to lentivirus infection, the culture preparation of the basal seed cell comprising: culturing the human immortalized liver cells to obtain the basal seed cells with the confluence rate of 50-70%.

3. The method of claim 1, wherein the packaged lentivirus is mixed with human immortalized hepatocytes at a ratio of: every 5X 10⁵Number of human immortalized hepatocytes and 1X 10⁷-2.5×10⁷A number of packaged lentivirus solutions were mixed.

4. The method of claim 3, wherein 48-72 hours after infection of the human immortalized hepatocytes, the subculture is continued by using a medium containing the screening agent; the generation number of the subculture is 3-4 generations.

5. The method of claim 4, wherein the screening agent is puromycin, phleomycin, blasticidin, hygromycin or neomycin.

6. The method of claim 1, wherein the liver-specific promoter is selected from the group consisting of a CMV promoter, a PGK promoter, an albumin promoter, an apolipoprotein E promoter, a phosphoenolpyruvate carboxykinase promoter, an alpha-I-antitrypsin promoter, a thyroid hormone binding globulin promoter, an alpha-fetoprotein promoter, an alcohol dehydrogenase promoter, an IGF-II promoter, a factor VIII promoter, an HBV basal core protein promoter, an HBV pre-s 2 protein promoter, a thyroxine-binding globulin promoter, a hybrid promoter of HCR-Ap0CII, an HCR-hAAT hybrid promoter, an AAT promoter bound to an enhancer element of a mouse albumin gene, a low density lipoprotein promoter, a pyruvate kinase promoter, a lecithin-cholesterol acyltransferase promoter, a mouse albumin gene enhancer element, a mouse albumin promoter, a mouse albumin gene enhancer element, a mouse protein promoter, a mouse protein, a cell, a protein, any one of an apolipoprotein H promoter, a transferrin promoter, a transthyretin promoter, promoters of alpha-fibrinogen and beta-fibrinogen, an alpha-I-antichymotrypsin promoter, an alpha-2-HS glycoprotein promoter, a haptoglobin promoter, a ceruloplasmin promoter, a plasminogen promoter, a complement protein promoter, a promoter of complement C3 activator, a hemopexin promoter, and an alpha-I-acidic glycoprotein promoter;

the first screening gene is selected from puromycin resistance gene, phleomycin resistance gene, blasticidin resistance gene, hygromycin resistance gene or neomycin resistance gene.

7. The method of claim 1, wherein the second selection gene is selected from the group consisting of eukaryotic resistance genes;

the eukaryotic resistance gene is selected from puromycin resistance gene, phleomycin resistance gene, blasticidin resistance gene, hygromycin resistance gene or neomycin resistance gene.

8. The method of claim 7, wherein said tetracycline-inducible promoter is selected from the TRE3G promoter.

9. Use of the Tet-on system for modulating transcription of hepatocytes according to claim 1 for the preparation of liver function-reversible, immortalized hepatocytes.

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