CN110540955B - Method for improving expression quantity of NROB2 gene in differentiated cell - Google Patents

Method for improving expression quantity of NROB2 gene in differentiated cell Download PDF

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CN110540955B
CN110540955B CN201910804586.XA CN201910804586A CN110540955B CN 110540955 B CN110540955 B CN 110540955B CN 201910804586 A CN201910804586 A CN 201910804586A CN 110540955 B CN110540955 B CN 110540955B
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孙浩
刘凤林
陈昱安
周云卿
蔡军
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Beijing Institute Of Collaborative Innovation
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    • 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
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/24—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the genital tract, from non-germinal gonad cells

Abstract

The invention provides a method for improving the expression quantity of NR0B2 gene in differentiated cells, belonging to the technical field of differentiation and preparation of regenerative cells. According to the invention, a liver cell generated by differentiation of a pluripotent stem cell is used for screening a lipid metabolism related micromolecule library, a micromolecule Int-747 capable of improving NR0B2 gene expression in the differentiated cell is identified, and the micromolecule is applied to the liver cell, so that the liver cell capable of highly expressing the NR0B2 gene is induced. The liver cells have high expression of NR0B2 gene, and considering the central action of NR0B2 in liver lipid/sugar metabolism, the differentiated cells with high expression of NR0B2 gene can be used for researching the lipid/sugar metabolism mechanism of liver and screening the drugs for regulating the lipid/sugar metabolism of the body.

Description

Method for improving expression quantity of NROB2 gene in differentiated cell
Technical Field
The invention belongs to the technical field of regenerative cell differentiation and preparation, and particularly relates to a method for improving the expression quantity of NROB2 gene in differentiated cells.
Background
Human pluripotent stem cells can be expanded indefinitely and differentiated to produce liver cells. Because the cell number is unlimited, the generated liver cells have very wide application prospects in the fields of disease models, drug screening, drug toxicity tests, tissue engineering, cell therapy, gene therapy and the like. Liver cells, which are currently differentiated from human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, are immature in cells, similar to fetal liver status. Accordingly, there was some difference in gene expression of the liver cells produced by differentiation and the mature liver cells (see FIG. 1).
Among these differentially expressed genes, a very important functional gene is the Nuclear Receptor gene NR0B2 (Nuclear Receptor subunit 0 Group B Member 2, also known as small heterodorimer partner, SHP). Gene expression of NR0B2 is regulated by NR1H4 (Nuclear Receptor subset 1 Group H Member 4, also known as Farnesoid X-Activated Receptor, FXR). The NR0B2 protein regulates the transcription activity of various nuclear receptors in cells and regulates the expression of target genes thereof. Nuclear receptor genes interacting with NR0B2 include Androgen Receptor (AR), Estrogen receptor (ESRa), Hepatocyte nuclear factor 4a (hepaticum nuclear factor 4a, HNF4 a), Liver receptor homolog 1 (Liver receptor homolog-1, LRH), Liver X receptor a (Liver X receptor alpha, LXRa), Peroxisome-activated receptor gamma (PPARg), Retinoic acid receptor alpha (RARa), and Retinoid X receptor (RXRa), and the like. Regulates the body's lipid/sugar metabolism, and is a central axis member that maintains the body's energy homeostasis.
In view of the important role of NR0B2 in liver lipid/carbohydrate metabolism, it is necessary to research and prepare a liver cell with high expression of NR0B2 to reduce the difference between the differentiated liver cell and the mature liver cell.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a liver cell with high NR0B2 content and a method for improving NROB2 gene in differentiated cells.
In order to achieve the purpose, the invention screens a small molecule library related to lipid metabolism, adds small molecules in the small molecule library into differentiated cells, and changes the liquid every two days. After 6 days of treatment, the cells were further differentiated to the final stage, sampled, RNA was extracted, and the expression level of NR0B2 gene in the cells was detected using RT-qPCR, thereby identifying small molecule Int-747 capable of increasing the expression of NR0B2 gene in the differentiated cells.
Int-747 (Obeticholic acid; 6-ECDCA; 6-ethylhepatolic acid) is a potent and selective Farnesoid X Receptor (FXR) agonist. It is known to prevent or even reverse liver fibrosis. Can be used as a human bile acid mimic to develop and treat PBC, non-alcoholic steatohepatitis and other liver diseases and intestinal diseases. The prior art does not find that Int-747 can improve the expression level of NR0B2 in liver differentiated cells.
In the present invention, based on the above findings, the present invention provides a method for increasing the expression level of NR0B2 gene in a differentiated cell, wherein Int-747 is added to a cell differentiation system at one or more stages of continued differentiation after endoderm cells are produced during cell differentiation.
Preferably, Int-747 is added to the cell differentiation system at the third or fourth stage of cell differentiation.
The differentiated cell is a cell generated by differentiation of a human pluripotent stem cell.
Preferably, the differentiated cell is a liver cell derived from differentiation of a human pluripotent stem cell.
Further, the present inventors have found through extensive studies that Int-747 is added to a cell differentiation system in an amount of 1 to 50. mu. mol/L, preferably 1 to 10. mu. mol/L.
The invention also provides a differentiated cell with high expression of the NR0B2 gene, which is prepared by the following method: during the process of cell differentiation, Int-747 is added into a cell differentiation system at one or more stages of continued differentiation after endoderm cells are produced, and cells with high expression of the NR0B2 gene are obtained after the differentiation is finished.
The differentiated cell is a liver cell generated by differentiation of the human pluripotent stem cell.
The differentiated cell with high expression of the NR0B2 gene and the differentiated cell obtained by the method can play a model role in preparing or screening medicines for regulating the fat or sugar metabolism of the organism, so the invention provides the application of the differentiated cell in preparing or screening the medicines for regulating the fat or sugar metabolism of the organism.
The invention provides application of the differentiated cell in a liver fat or sugar metabolism model.
Furthermore, the invention provides an application of Int-747 in improving the expression level of NR0B2 gene in liver differentiated cells.
According to the invention, a liver cell generated by differentiation of a pluripotent stem cell is used for screening a lipid metabolism related micromolecule library, a micromolecule Int-747 capable of improving NR0B2 gene expression in the differentiated cell is identified, and the micromolecule is applied to the liver cell, so that the liver cell capable of highly expressing the NR0B2 gene is induced. The liver cells have high NR0B2 gene expression, and due to the central action of NR0B2 in liver lipid/sugar metabolism, the differentiated cells of the high-expression NR0B2 gene can be used for researching the lipid/sugar metabolism mechanism of the liver and screening drugs for regulating the lipid/sugar metabolism of an organism, have excellent clinical application value and have good application prospect in the field of drug research and development.
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FIG. 1 shows RNA-Seq data for gene expression in differentiated cells and liver cells showing low expression of the NR0B2 gene in differentiated cells.
FIG. 2 is a schematic diagram of the screening process of example 2.
FIG. 3 is a typical screening result of example 2. In the figure, the ordinate is relative gene expression amount, the abscissa is position detected by each small molecule, that is, fig. 3 is relative amount of each small molecule to NR0B2 gene expression, and positions 3-a4 are added with lnt-747, and the expression amount is very obvious.
FIG. 4 shows that Int-747 can increase the expression of NR0B2 gene in differentiated cells. The left graph is ALB marker protein of normal liver cells, and it can be seen that the differentiation efficiency of three cells is not obviously different; the right panel shows that the expression of the differentiation efficiency of the NR0B 2-labeled protein was increased by the genetically modified cells, and the differentiation efficiency was increased by hundreds of times when the amount of int747 was added by 10. mu. mol and when no int747 was added (i.e., only the solvent DMSO).
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the embodiment of the invention, human induced pluripotent Stem Cell iPS18 (purchased from Clonetech) is taken as an example, and a liver differentiation scheme is modified from Cai J et al, Protocol for directed differentiation of human pluripotent Stem cells heated a hepatocyte sulfate, StemBook Harvard Stem Cell Institute, 2008. The small molecule library is screened in the third stage of differentiation, and the gene expression is detected in the end of the fourth stage of differentiation. The screening strategy is schematically shown in FIG. 2, and representative screening results are shown in FIG. 3. The embodiment of the invention is only used as an example to illustrate the effect of Int-747 on improving the expression of NR0B2 gene in differentiated cells (see FIG. 4), and can be applied to other systems for differentiating iPS into liver cells in practical application, including but not limited to using different iPS cell lines or ES cell lines, adopting different differentiation strategies, using different concentrations and acting durations, and the like. The reagents and materials used in the scheme are commercial products, and products from various sources have the same effect.
Example 1 iPS cell recovery, culture and passaging
Coating Geltrex: geltrex (Thermo Fisher Scientific) was thawed on ice and then diluted to 2 mg/ml with ice cold DF12 (Thermo Fisher Scientific). 5 ml of the solution was added to a 10 cm dish and left at 37 ℃ for 30 minutes to 4 hours. Formulation PGM 1: PGM1 medium was purchased from Seebia, and the supplement was thawed at 4 ℃ and added to the basal medium to obtain PGM1 medium. 12 ml of PGM1 was taken and 5. mu.M of small molecule Y27632 (Selleck) was added. The water bath was heated to 37 ℃. The cryopreserved iPS cells were removed, purchased from clonetech, quickly placed in a water bath, gently shaken, and the ice cubes were known to be completely thawed. And (5) wiping the frozen tube. The cells were gently transferred to a 15 ml centrifuge tube. 5 ml of PGM1 containing Y27632 was added dropwise with gentle shaking. Centrifuge at 1000 rpm for 3 minutes. The supernatant was discarded, 7 ml of PGM1 containing Y27632 was added, and the cells were resuspended. The Geltrex coated petri dish was removed and the supernatant was aspirated. The cells were seeded into a petri dish and the dish was shaken to evenly distribute the cells in the dish. The cells were cultured at 37 ℃ in a cell culture chamber containing 5% CO 2. Cells were replaced daily. After 4-5 days, cells were passaged when they had risen to 80% -90% confluence.
The supernatant of the cell dish to be passaged was aspirated and washed once with DPBS. 3 ml of Versene (Thermo Fisher Scientific) was added and left at 37 ℃ for 2 minutes. Versene was discarded and cells were gently blown down with PGM1 medium containing 5. mu. M Y27632. Cells from 1/10-1/15 were plated onto Geltrex coated dishes. 7 ml of PGM1 medium containing Y27632 was added to each dish. The dish was shaken to distribute the cells evenly in the dish and returned to the 37 ℃ incubator for at least 2-3 hours without movement.
Example 2 differentiation of iPS cells into liver cells and Small molecule screening
The screening scheme is schematically shown in FIG. 2. After 4-5 days of passage, when iPS cells grow to 80% -90% confluence, the iPS cells are digested by AccutaseTo 3X 106Wells were seeded into Geltrex coated 6-well plates; after about 18 hours, wash once with 1640 medium, then change to 1640 + 2% B27 (Insulin free, from Thermo-Fisher) + 100 ng/ml Activin A (Peprotech) + 3. mu.M CHIR99021 (Selleck); after 24 hours, the medium was changed to 1640 + 2% B27 (without Insulin) + 100 ng/ml Activin A. At this time, a great deal of cell death occurs, which is a normal phenomenon. If too many dead cells are present, they can be washed with 1650 medium and replaced. Changing the liquid every day for two days; after 48 hours, on the third day of differentiation, differentiated cells were digested with Accutase at 5X 105Wells were seeded into Geltrex coated 24-well plates; the adherent culture medium is: 1640 + 2% B27 + 20 ng/ml BMP4 + 10 ng/ml bFGF + 5. mu. M Y-27632; after 24 hours, the cells were attached and the medium was changed to 1640 + 2% B27 + 20 ng/ml BMP4 + 10 ng/ml bFGF. Changing the liquid every two days for 5 times; after 5 days, the medium was changed to 1640 + 2% B27 + 1/10000 volumes of small molecules. Small molecules were from the small molecule library L-7000 (from the ceramic Biochemical company) and controls were DMSO of the same volume. Changing the liquid once every 2 days, and changing for 3 times; after 6 days, the medium was changed to HCM + 20 ng/ml OSM. The liquid was changed once a day, 5 times. After 5 days, the cells were sampled, analyzed by quantitative PCR assay, and the results of the assay are shown in FIG. 3, which shows the effect of each small molecule on the NR0B2 gene, wherein the effect of the small molecule Int-747 is the largest (highest position of the ratio column in the figure), and the differentiation efficiency at the addition of 10. mu. mol is hundreds of times higher than that without the addition of Int 747. As can be seen from fig. 3, the present invention can reach 0.58 calculated as the relative expression level of mature hepatocytes 1, but only at a level of 0.0X without the addition of int 747.
Example 3 RNA preparation and RT-qPCR
Cells were lysed with Trizol (Thermo-Fisher) and RNA was extracted using an RNA extraction kit (Zymo). The OD260/230 of the RNA is >1.5, and the OD260/280 is between 1.95 and 2.05. Mu.g of RNA was taken and reverse-transcribed (Promega kit) to 20. mu.l of cDNA, which was then diluted to 100. mu.l, and the gene expression level was detected by qPCR mix (Applied system) in a qPCR instrument (Nordheim source). The qPCR primer is synthesized by the phylogenetic department, and the product size is between 120 and 250 bp. ALB is a protein used to measure differentiation efficiency. The primer sequences used for PCR were:
TABLE 1 qPCR primer sequences
Figure 686485DEST_PATH_IMAGE001
The PCR reaction system is as follows: mu.l of cDNA, 0.2. mu.l of each primer (10. mu.M), 2.6. mu.l of water, and 5. mu.l of qPCR master mix.
The PCR reaction conditions are as follows: 950 C 10 min；95 0 C 15 sec，60 0C60 sec, 40 cycles.
And obtaining the Ct value of the amplification. Using GAPDH as an internal control, the expression of the corresponding gene in liver tissue was set to 1.
By adding int747 to the differentiation system, the expression of NR0B2 gene by differentiation was increased hundreds of times, as shown in fig. 4. The expression quantity of NR0B2 is made to approach to that of normal human liver cell, so as to improve the immature condition of lipid metabolism and carbohydrate metabolism of differentiated liver cell obtained in the prior art and raise the cell differentiation quality.
The hepatic cells differentiated by the invention have better lipid metabolism and carbohydrate metabolism performance, the hepatic cells differentiated by the hepatic cells and the common protein A interventional differentiation mode are detected by a glucose oxidation method, the hepatic cells are cultured in 1.0g/L glucose culture solution for 24h to detect the content of glucose in the culture solution, and under the condition of the same initial concentration, the glucose concentration in the cell culture solution is obviously 0.45 g/L, and the glucose concentration in the hepatic cell culture solution differentiated by the common protein A interventional method is 0.69 g/L. It is evident that the cells differentiated by the method of the invention have a better capacity for sugar metabolism.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (5)

1. A method for increasing the expression level of NR0B2 gene in differentiated liver cells, comprising adding Int-747 to a cell differentiation system at one or more stages of continued differentiation after endoderm cell generation during cell differentiation;
wherein, the differentiated liver cell is a liver cell produced by a human pluripotent stem cell;
wherein the one or more stages of continued differentiation after endoderm cells are at day 5 of cell differentiation, Int-747 is added to the cell differentiation system in an amount of 1-10 μmol/L.
2. A differentiated liver cell highly expressing an NR0B2 gene, which is prepared by the following method: adding Int-747 into a cell differentiation system at one or more stages of continuous differentiation after endoderm cells are generated in the process of cell differentiation, and obtaining cells with high expression of NR0B2 gene after the differentiation is finished; wherein, the differentiated liver cell with high expression of the NR0B2 gene is a liver cell generated by the differentiation of a human pluripotent stem cell with high expression of the NR0B2 gene;
wherein the one or more stages of continued differentiation after endoderm cells are at day 5 of cell differentiation;
int-747 is added into the cell differentiation system in an amount of 1-10. mu. mol/L.
3. Use of the differentiated liver cell of claim 2 or the cell produced by the method of claim 1 for the preparation or screening of a drug for regulating fat or sugar metabolism in the body.
4. Use of the differentiated liver cell of claim 2 or the cell produced by the method of claim 1 as a model of liver fat or sugar metabolism.
Use of Int-747 for increasing the expression level of NROB2 gene in liver differentiated cells, wherein Int-747 is added to the liver cell differentiation system during the course of liver cell differentiation at one or more stages of continued differentiation after endoderm cells have been produced;
wherein the one or more stages of continued differentiation after endoderm cells are at day 5 of liver cell differentiation, Int-747 is added to the liver cell differentiation system in an amount of 1-10 μmol/L.
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WO2000037077A1 (en) * 1998-12-23 2000-06-29 Glaxo Group Limited Assays for ligands for nuclear receptors
JP2005281155A (en) * 2004-03-29 2005-10-13 Japan Health Science Foundation Cholesterol homeostasis-related gene transfer activity modifier via fxr activation
CN102858958A (en) * 2010-02-03 2013-01-02 日本国立癌症研究中心 Induced Hepatic Stem Cell And Process For Production Thereof, And Applications Of The Cell
CN105377870A (en) * 2013-05-14 2016-03-02 英特塞普特医药品公司 11-hydroxyl-derivatives of bile acids and amino acid conjugates thereof as farnesoid x receptor modulators
CN106518708A (en) * 2016-07-18 2017-03-22 中国药科大学 Synthesis and applications of novel FXR agonists
CN109303921A (en) * 2018-10-30 2019-02-05 中国药科大学 FXR agonist and the combination of SIRT1 agonist are preparing the application in anti-hepatic fibrosis medicines

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037077A1 (en) * 1998-12-23 2000-06-29 Glaxo Group Limited Assays for ligands for nuclear receptors
JP2005281155A (en) * 2004-03-29 2005-10-13 Japan Health Science Foundation Cholesterol homeostasis-related gene transfer activity modifier via fxr activation
CN102858958A (en) * 2010-02-03 2013-01-02 日本国立癌症研究中心 Induced Hepatic Stem Cell And Process For Production Thereof, And Applications Of The Cell
CN105377870A (en) * 2013-05-14 2016-03-02 英特塞普特医药品公司 11-hydroxyl-derivatives of bile acids and amino acid conjugates thereof as farnesoid x receptor modulators
CN106518708A (en) * 2016-07-18 2017-03-22 中国药科大学 Synthesis and applications of novel FXR agonists
CN109303921A (en) * 2018-10-30 2019-02-05 中国药科大学 FXR agonist and the combination of SIRT1 agonist are preparing the application in anti-hepatic fibrosis medicines

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