CN111621546A - Method for screening thyroxine receptor agonist by using primary hepatocytes - Google Patents

Method for screening thyroxine receptor agonist by using primary hepatocytes Download PDF

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CN111621546A
CN111621546A CN202010557348.6A CN202010557348A CN111621546A CN 111621546 A CN111621546 A CN 111621546A CN 202010557348 A CN202010557348 A CN 202010557348A CN 111621546 A CN111621546 A CN 111621546A
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thyroxine
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receptor agonist
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primary hepatocytes
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徐剑锋
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Shanghai Chengyi Biotechnology Co ltd
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Abstract

The invention discloses a method for screening a thyroxine receptor stimulant by utilizing primary hepatocytes. The method comprises the following steps: step 1, constructing a vector plasmid TRE-RG with a reporter gene and a thyroid hormone response element; and 2, packaging the vector plasmid TRE-RG into TRE-luc recombinant adenovirus by using adenovirus, infecting primary hepatocytes with the recombinant adenovirus, adding a compound to be tested, and determining whether the compound to be tested is a thyroxine receptor agonist according to an expression product of a reporter gene. The invention creatively utilizes the height of primary hepatocytes to express the thyroxine receptor, only needs to clone a small section of highly conserved thyroxine reaction element for constructing a reporter gene vector, avoids the difficult problem of cloning the whole thyroxine receptor or the gene sequence of the Ligand Binding Domain (LBD) of the thyroxine receptor, can be applied to primary hepatocytes of different species, and increases the application flexibility. The method is simple and high in infection efficiency, and solves the problems of low transfection efficiency, poor repeatability and poor experimental stability of the existing method.

Description

Method for screening thyroxine receptor agonist by using primary hepatocytes
Technical Field
The invention belongs to the technical field of drug screening, and particularly relates to a method for screening a thyroxine receptor agonist by using primary hepatocytes.
Background
Thyroid hormone receptor (TR) agonists have great therapeutic potential in a range of metabolic diseases, and the application fields include but are not limited to obesity, hyperlipidemia, thyroid disease, alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic steatosis, hepatic fibrosis, hypercholesterolemia, familial hypercholesterolemia (HeFH/HoFH), X-associated adrenoleukodystrophy (X-ALD), diabetes, atherosclerosis, hypertension, coronary heart disease, depression, osteoporosis, arrhythmia, congestive heart failure and other diseases. Early thyroid hormone receptor agonists have hindered clinical development due to side effects in the cardiovascular and skeletal systems. In recent years, liver-targeted thyroid hormone receptor agonists such as MGL3196, VK2809, etc., have demonstrated beneficial therapeutic effects of thyroid hormone receptor agonists in clinical trials for treating NASH and hypercholesterolemia, such as lowering low density lipoproteins and raising high density lipoproteins, promoting cholesterol metabolism to bile acids, lowering triglycerides, while avoiding side effects of thyroid hormone on cardiac function (tachycardia, increased stroke volume, increased cardiac index, myocardial hypertrophy, decreased peripheral vascular resistance, increased pulse pressure) and skeletal system (osteoporosis), demonstrating that this drug development protocol is feasible and has great potential. Therefore, screening thyroid hormone receptor agonists with liver selectivity is crucial for the development of this class of drugs.
In the prior art, screening systems for thyroid hormone receptor agonists mainly comprise a cell screening system of HEK293 transient transthyretin receptors and a cell screening system of HEK293 stable transthyretin receptors. There are four main methods for introducing foreign genes into cells: electrical shock, calcium phosphate, liposome-mediated, and virus-mediated methods. The electric shock method is to temporarily perforate the cell for a short time to allow the foreign plasmid to enter; the calcium phosphate method and the liposome method are that different carrier substances carrying plasmids are utilized to make exogenous genes enter cells by a method of direct membrane penetration or membrane fusion; but because the experimental conditions of the electric shock method and the calcium phosphate method are controlled more tightly and the difficulty is higher; the prior preparation of the conventional virus method is complex and can have great influence on cells; therefore, for many common cell lines, the general transient transfection method mostly adopts a liposome method to transfer exogenous genes such as plasmids of thyroxine receptor genes into HEK293 cells, and uses intracellular mechanisms to express the thyroxine receptors. The common transthyretin receptor has low transfection efficiency, poor repeatability and poor experimental stability, a plurality of cell lines are difficult to transfect successfully, the toxicity of a common liposome transfection reagent is high, and transfection conditions such as the ratio of liposome to plasmid, the cell density, the transfection time and the content of serum in a culture medium need to be optimized and improved through a plurality of experimental groping. The cell system of HEK293 stable transthyretin receptor is a method for selecting a transfected single cell which can stably inherit a thyroxine receptor gene and can not be rapidly lost along with the increase of cell culture algebra on the basis of the cell system of HEK293 transient transthyretin receptor, and rapidly proliferating the single cell to meet the requirement of drug screening. The establishment period of the transthyretin receptor system is long, and the HEK293 cell is a non-liver cell line, and if a liver selective drug is screened, the specific transporter and metabolic function of the liver cell cannot be simulated.
Abbreviations and key term definitions in the present invention:
TR, thyroid hormone receptor
TRE, Throid Hormone responses thyroxine reactive element
NAFLD, non-alcoholic fatty liver disease
NASH, Non-alcoholic steatohepatitis, nonalcoholic steatohepatitis
HeFH/HoFH, Heterozygous/Homozygous Familial hypercholesterolaemia Heterozygous/Homozygous Familial Hypercholesterolemia
X-ALD, X-linked adrenoleukodystrophy LBD, ligand binding domain
PCR, Polymerase Chain Reaction of Polymerase Chain Reaction
Luc, luciferase
Beetle luciferin (Beetl luciferin)
AMP, adenosine monophosphate
Disclosure of Invention
The invention aims to provide a method for screening a thyroxine receptor agonist by using primary hepatocytes. Mainly solves the problems that the screening system in the prior art has low transfection efficiency and high cost and can not simulate the specific transporter and metabolic function of liver cells.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for screening a thyroxine receptor agonist by using primary hepatocytes comprises the following steps:
step 1, constructing a vector plasmid TRE-RG with a reporter gene and a thyroid hormone response element;
and 2, packaging the vector plasmid TRE-RG into TRE-luc recombinant adenovirus by using adenovirus, infecting primary hepatocytes with the obtained recombinant adenovirus, adding a compound to be detected, and determining whether the compound to be detected is a thyroxine receptor agonist and the agonistic activity of the thyroxine receptor agonist according to a chemiluminescent signal generated by a chemical reaction of an expression product of the reporter gene and a substrate.
As a preferred embodiment, the reporter gene is a luciferase reporter gene, or a green fluorescent protein gene.
As a preferred embodiment, the sequence of the thyroid hormone responsive element is as shown in SEQ ID NO. 1.
As a preferred embodiment, the sequence of the thyroid hormone response element can adopt any one of the following sequences or 1-10 times of repetition and combination of any one of the following sequences:
(1) 5-AGGTCANNNNAGGTCA-3 of the direct repetitive sequence;
(2) palindromic sequence 5-AGGTCATGACCT-3;
(3) reverse palindromic sequence 5-TGACCTNNNNNNAGGTCA-3;
n in the sequence represents A, T, G, C.
The invention adopts 4 TRE sequences with same direction repetition to be connected in series to obtain a sequence shown in SEQ ID NO.1 to construct a vector plasmid TRE-RG, the thyroid hormone reaction element can be replaced by any one of the sequences or repeated and combined for 1 to 10 times of the sequences, and the repetition and combination can be repeated and combined by one of the sequences or combined by one of the sequences and other sequences.
As a preferred embodiment, the TRE-luc recombinant adenovirus is assembled by: and (3) transferring the carrier plasmid TRE-RG and the virus backbone plasmid into 293 series cells for assembly to obtain TRE-luc recombinant transfection virus.
As a preferred embodiment, the 293 series of cells are 293 cells, HEK293A cells, HEK293T cells, and other cell lines for packaging adenovirus, lentivirus, or adeno-associated virus.
In a preferred embodiment, the recombinant transfected virus is a recombinant adenovirus, a recombinant lentivirus, or a recombinant adeno-associated virus.
As a preferred embodiment, the primary hepatocyte is a rat primary hepatocyte, a mouse primary hepatocyte, a human primary hepatocyte, a guinea pig primary hepatocyte, a dog primary hepatocyte, a pig primary hepatocyte, a rabbit primary hepatocyte or a monkey primary hepatocyte.
As a specific embodiment, the present invention provides a method for screening for a thyroxine receptor agonist on rat primary hepatocytes based on a luciferase reporter gene system. The method comprises the steps of firstly constructing a carrier plasmid with a luciferase reporter gene and a Thyroxine Reaction Element (TRE), then transferring the carrier plasmid and virus skeleton plasmid into 293 cells for assembly to obtain recombinant adenovirus, infecting primary rat hepatocytes with the assembled virus with the luciferase reporter gene and the thyroxine reaction element, reacting luciferase with substrate luciferin, and detecting a chemiluminescent signal by using a microplate reader, so that the condition that a compound to be screened activates a thyroxine receptor can be reflected.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively utilizes the height of primary hepatocytes to express the thyroxine receptor, only needs to clone a small section of highly conserved thyroxine reaction element for constructing a reporter gene vector, avoids the difficult problem of cloning the whole thyroxine receptor or the gene sequence of the Ligand Binding Domain (LBD) of the thyroxine receptor, can be applied to the primary hepatocytes of different species, and increases the application flexibility.
2, the method adopts adenovirus infection, is simple and has high infection efficiency, and solves the problems of low transfection efficiency, poor repeatability and poor experimental stability of the existing method.
3, the method can evaluate the activation condition of the thyroxine receptor of the compound with high flux, rapidness and high efficiency on the level of primary hepatocytes of rats, and has simple steps and good stability.
Drawings
FIG. 1 is a graph showing the dose-response activity of different concentrations of T3 and MGL3196 in the present invention on adenovirus vector-infected rat primary hepatocytes.
Detailed Description
The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified.
The thyroxine receptor forms heterodimers with the retinoid receptor (RXR) and modulates gene expression through interaction with thyroxine-responsive elements (TRE) and various nuclear co-activators (coactivators) and nuclear co-inhibitors (corepressors). The thyroxine receptor forms heterodimer with RXR, binds to the Thyroxine Response Elements (TREs) in the promoter region of a target gene, and when the thyroxine receptor does not bind to T3(T3 is an activated endogenous ligand of the thyroxine receptor), the complex formed by co-inhibited transcription proteins HDAC3, TBL1 and NcoR/SMART is recruited to inhibit the transcription of downstream genes and is in a resting state. When the thyroxine receptor binds to the T3 ligand, nuclear co-activators (coactivors) SRC-1/p160, CBP/p300, TRAPs, which form a complex with RXR, are recruited to activate transcription of downstream genes. Hair brushThe reporter gene of luciferase is connected after TRE, when T3 or T3 analogue such as candidate compound and primary liver cell are incubated together, the candidate compound binds to thyroxine receptor, then transcription is activated, luciferase is transcribed and expressed, and when luciferin substrate of luciferase is added, luciferase utilizes ATP and O in cell in the presence of magnesium ion2Catalyzing the luciferin reaction to generate Oxyluciferin, AMP, PPi, CO2And a biochemical luminescence phenomenon is generated, so that the activation condition of the thyroxine receptor can be measured by detecting the light intensity of the luminescences by using a microplate reader.
EXAMPLE 1 construction of the thyroid hormone response element and the luciferase reporter vector plasmid TRE-luc
pHBAD-EF1-MCS-3flag-CMV-LUC- △ loxp vector (purchased from Henan Han dynasty organism, catalog number HBAD-1016) is subjected to double digestion by XbaI/NheI, and after the digestion of the vector is finished, DNA gel recovery and purification kit (column centrifugation type) is used for recovering, a sequence (shown as SEQ ID NO. 2) containing a thyroid hormone reaction element and a luciferase reporter gene promoter is synthesized, namely the sequence shown as SEQ ID NO.2 contains two parts of a sequence SEQ ID NO.1 and a reporter gene promoter sequence, the synthesized fragment is subjected to PCR amplification, and then the sequence is directly cloned onto pHBAD-EF1-MCS-3flag-CMV-LUC- △ loxp empty vector, and competent cells DH5a, CaCl and C are used2Carrying out process transformation, selecting ampicillin (ampicillin) resistant bacteria, shaking the bacteria at 37 ℃ at 250 rpm, carrying out PCR identification on the bacteria liquid, sending the positive clone bacteria liquid to a sequencing company for sequencing, comparing the positive clone bacteria liquid with a target sequence, and completing construction if the result sequence is consistent to obtain TRE-luc vector plasmid.
EXAMPLE 2 Assembly of recombinant adenovirus vectors
And (3) when the 293 cell grows to 70-80% of the basal area, transfecting the prepared TRE-luc vector plasmid and the skeleton plasmid pHBAd-BHG by using a LipofiterTM transfection reagent. After 6 hours of transfection, the cell culture medium was replaced with fresh one. And (3) when most cells are diseased and fall off from the bottom, performing virus collection, repeatedly freezing and thawing for three times in liquid nitrogen and 37 ℃ water bath, centrifuging for 5 minutes at 3000rpm, collecting virus-containing supernatant, and discarding the precipitate. The supernatant is the TRE-luc first generation virus (P1) and will be used as the virus for subsequent large-scale virus amplification. And repeating the amplification and virus collection of each generation later, obtaining a large amount of TRE-luc recombinant viruses by amplification, purifying by a two-step CsCl ultracentrifugation method, further purifying by a dialysis method, namely dialyzing three times by using dialysis buffer solution with the volume of 200 multiplied by one time at one-hour interval, detecting the infectious titer by adopting an improved TCID50 method after the dialysis is finished, packaging when the titer meets the requirement, and storing at-80 ℃ for later use.
Example 3 screening experiments for Virus-infected rat Primary hepatocytes and thyroxine receptor agonists
The liver of an SD rat with the age of 5-10 weeks is perfused in situ by using D-Hanks liquid and collagenase digestive liquid in two steps, the purified liver cells are obtained after the cells are separated and are subjected to low-speed centrifugation (50 Xg, 5min) and Percoll gradient centrifugation, and the liver cells with the survival rate of more than 90 percent are used for the next experiment.
Counting rat primary hepatocytes, diluting to cell sap with cell number of 400,000/mL, inoculating 50ul of the cell sap into a collagen pre-coated 96-well plate, incubating at 37 ℃ with 5% CO2Culturing for at least 1 h. A mixture of 50 or 100MOI of the TRE-luc recombinant adenovirus prepared in example 2 and 16. mu.g/mL polybrene (Sigma #107689) was diluted, and 8 concentration-gradient test compounds T3 and MGL3196(T3 is a natural thyroid receptor agonist and MGL3196 is a liver-targeted thyroid receptor agonist used in phase 3 clinical trials) were diluted with the mixture. Adding the mixed solution containing recombinant adenovirus and the compound to be tested at 50 ul/well, centrifuging at 1000g for 15min at 37 deg.C and 5% CO2After overnight incubation for 24h, 25. mu.L/well of Steady-Glo luciferase assay reagent (Promega, cat. No. E2520) was added, shaken on a shaker for 10min, and the intensity of chemiluminescence (luminescence) was measured by a multifunctional microplate reader.
Activity calculation of test compounds: the thyroid receptor agonistic effect at a maximum concentration of 2000nM of T3 was set to 100%, i.e., the activity of the test compound relative to T3 was set to 100% × (test compound reading-blank DMSO reading)/(maximum concentration of 2000nM reading-blank DMSO reading of T3), and the maximum effect (Emax) and half-maximum effect concentration (EC50) were obtained by four-parameter logistic curve fitting. Referring to fig. 1: wherein FIG. 1 is a graph showing the dose-response activity of different concentrations of T3 and MGL3196 on adenovirus vector-infected rat primary hepatocytes. As can be seen from the data results of fig. 1: as the treatment concentration of T3 and MGL3196 increased, the percentage of relative chemiluminescent signal from the luciferase reaction with the substrate also increased accordingly. This effect is dose dependent, with Emax and EC50 as shown in table 1.
TABLE 1
Compound (I) EC50(nM) Emax(％)
T3 7.5 100
MGL3196 727.7 77
From the above data, it can be seen that: treatment with T3 and MGL3196 resulted in the expression of the reporter gene luciferase, both of which were dose-dependent upregulation of reporter gene production in adenovirus vector-infected rat primary hepatocytes. Therefore, whether the compound to be tested is the thyroxine receptor stimulant can be determined through the expression quantity of the reporter gene luciferase, so that not only can the screening of the thyroxine receptor stimulant be realized, but also the activity ranking of the thyroxine receptor agonized by the compound to be tested can be realized, and the thyroxine receptor stimulant with the highest activity and selectivity of liver cells can be screened.
The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.
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Claims (8)

1. A method for screening a thyroxine receptor agonist by using primary hepatocytes comprises the following steps:
step 1, constructing a vector plasmid TRE-RG with a reporter gene and a thyroid hormone response element;
and 2, assembling the vector plasmid TRE-RG into TRE-luc recombinant adenovirus by using adenovirus, infecting primary hepatocytes with the obtained recombinant adenovirus, adding a compound to be tested, and determining whether the compound to be tested is a thyroxine receptor agonist and the strength of the agonist activity of the thyroxine receptor agonist according to related signals of a reporter gene expression product.
2. The method of screening for a thyroxine receptor agonist using primary hepatocytes as recited in claim 1 wherein: the reporter gene is a luciferase reporter gene or a green fluorescent protein gene.
3. The method of screening for a thyroxine receptor agonist using primary hepatocytes as recited in claim 1 wherein: the sequence of the thyroid hormone response element is shown as SEQ ID NO. 1.
4. The method of screening for a thyroxine receptor agonist using primary hepatocytes as claimed in claim 1, wherein the sequence of the thyroid hormone responsive element is selected from any of the following sequences or from 1 to 10 repeats and combinations of any of the following sequences:
(1) 5-AGGTCANNNNAGGTCA-3 of the direct repetitive sequence;
(2) palindromic sequence 5-AGGTCATGACCT-3;
(3) reverse palindromic sequence 5-TGACCTNNNNNNAGGTCA-3;
n in the sequence represents A, T, G, C.
5. The method of screening for a thyroxine receptor agonist using primary hepatocytes as claimed in claim 1, wherein the TRE-luc recombinant adenovirus is assembled by: and (3) transferring the carrier plasmid TRE-RG and the virus backbone plasmid into 293 series cells for assembly to obtain TRE-luc recombinant transfection virus.
6. The method of screening for a thyroxine receptor agonist using primary hepatocytes as recited in claim 1 wherein: the 293 series cells are 293 cells, HEK293A cells, HEK293T cells and other cell lines for packaging adenovirus, lentivirus or adeno-associated virus.
7. The method of screening for a thyroxine receptor agonist using primary hepatocytes as recited in claim 1 wherein: the recombinant transfection virus is a recombinant adenovirus, a recombinant lentivirus or a recombinant adeno-associated virus.
8. The method of screening for a thyroxine receptor agonist using primary hepatocytes as recited in claim 1 wherein: the primary hepatocyte is rat primary hepatocyte, mouse primary hepatocyte, human primary hepatocyte, guinea pig primary hepatocyte, dog primary hepatocyte, pig primary hepatocyte, rabbit primary hepatocyte or monkey primary hepatocyte.
CN202010557348.6A 2020-06-18 2020-06-18 Method for screening thyroxine receptor agonist by using primary hepatocytes Pending CN111621546A (en)

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MARTIN A. PAQUETTE等: "（"Thyroid Hormone Response Element Half-Site Organization and Its Effect on Thyroid Hormone Mediated Transcription"", 《PLOS ONE》 *
郭志兰等: ""F-κB荧光素酶报告基因系统的构建及验证"", 《生物工程学报》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114437202A (en) * 2022-01-18 2022-05-06 四川大学华西医院 Androgen response element and application thereof in detecting environmental androgen

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