阅读说明：本技术 一种利用原代肝细胞筛选甲状腺素受体激动剂的方法 (Method for screening thyroxine receptor agonist by using primary hepatocytes ) 是由 徐剑锋 于 2020-06-18 设计创作，主要内容包括：本发明公开了一种利用原代肝细胞筛选甲状腺素受体激动剂的方法。该方法为：步骤1,构建带有报告基因和甲状腺激素反应元件的载体质粒TRE-RG；步骤2,将所述载体质粒TRE-RG用腺病毒包装成TRE-luc重组腺病毒,在将此重组腺病毒感染原代肝细胞,加入待测化合物,根据报告基因的表达产物确定待测化合物是否为甲状腺素受体激动剂。本发明创造性地利用原代肝细胞本身高表达甲状腺素受体,仅需克隆一小段高度保守的甲状腺素反应元件用于构建报告基因载体,避免了克隆整个甲状腺素受体或者甲状腺素受体的配体结合区域(LBD)的基因序列的难题,同时可以应用于不同种属的原代肝细胞,增加了应用灵活性。本发明方法简单,感染效率高,克服了现有方法转染效率低,重复性差,实验稳定性差的问题。(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.)

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.

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.

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.