CN110904047B

CN110904047B - Cell model for screening developmental toxicity exogenous compound by taking 11 beta-HSD 2 as target spot, construction method and application thereof

Info

Publication number: CN110904047B
Application number: CN201911166168.9A
Authority: CN
Inventors: 汪晖; 陈廖斌; 徐丹; 文印宪; 齐勇建; 李斌; 张棋
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2022-01-04
Anticipated expiration: 2039-11-25
Also published as: CN110904047A

Abstract

The invention provides a cell model for screening developmental toxic exogenous compounds by taking 11 beta-HSD 2 as a target spot, a construction method and application thereof. The invention adopts human WJ-MSCs as target cells, and the luciferase reporter gene containing 11 beta-HSD 2 promoter is transfected into the target cells to obtain the cell model, and the model has two indexes: 11 β -HSD2 gene expression and luciferase activity of the reporter system. By using the model of the invention, the 11 beta-HSD 2 gene expression and luciferase activity of the test compound treatment group are reduced, which indicates that the test substance has developmental toxicity. The developmental toxicity exogenous compound screening cell model established by the invention has the characteristics of high specificity, high sensitivity, high stability, high feasibility and the like, can be used for high-throughput screening, and has important significance for rapidly screening exogenous compounds with developmental toxicity.

Description

Cell model for screening developmental toxicity exogenous compound by taking 11 beta-HSD 2 as target spot, construction method and application thereof

Technical Field

The invention belongs to the field of drug toxicology, and relates to a cell model for screening and developing toxic exogenous compounds by taking 11 beta-HSD 2 as a target spot, a construction method and application thereof.

Background

The developmental toxicity of the exogenous compound refers to structural or functional damage of offspring from intrauterine to pre-sexual maturity caused by the exogenous compound, and comprises structural deformity, dysfunction, growth retardation and even death. Developmental toxicity is one of the important matters for safety evaluation of exogenous compounds. Most of the existing evaluation means take the whole animal reproduction experiment result as a measure index of the development toxicity of the exogenous compound, but have the defects of large animal dosage, long experiment period, poor specificity, low sensitivity, incapability of clarifying the generation mechanism from the cellular or molecular level and the like. Even developmental toxicity in vitro evaluation systems developed gradually in recent years, such as whole embryo culture, micelle culture and embryonic stem cell culture, have the problems of high culture difficulty, high cost, limited time, low specificity of detection indexes and the like. In conclusion, the exogenous compound developmental toxicity in-vitro evaluation system has great limitations and is mainly related to unclear exogenous compound developmental toxicity expression and lack of a generation mechanism theoretical system, so that a high-throughput exogenous compound developmental toxicity in-vitro evaluation system based on a toxicity mechanism and a molecular target is yet to be developed.

Basal Glucocorticoid (GC) levels are critical in determining morphology and functional maturation of tissues in the uterine tube, and excessive GC exposure can cause toxicity in development of multiple organs. It is known that GC action on fetal tissues depends not only on circulating GC levels but also on the expression of 11 β -hydroxysteroid dehydrogenase (11 β -HSD) that mediates GC metabolism in fetal tissues. 11 β -HSD2 effectively regulates the locally active GC levels in fetal tissues by metabolically inactivating GC and is an important regulatory point for protecting the fetus from maternal GC interference. In the early stage, the inventor proposes and systematically verifies that the offspring rat multi-organ development toxicity caused by the exposure of the exogenous substance in the pregnancy period and the susceptibility of the adult chronic disease are the neuroendocrine metabolism programming mechanism for the first time internationally. Recently, we further propose that the development toxicity of offspring multi-organ caused by the exposure of exogenous compound during pregnancy is related to the over-exposure of maternal GC and the low expression of 11 beta-HSD 2, which is an important gene related to the local development of offspring tissues. It is suggested that 11 β -HSD2 may be a key target for mediating multi-organ developmental toxicity.

With the development of biotechnology, people have increasingly recognized fetal diseases, and a screening method based on specific molecular targets replaces the traditional non-specific method. A prediction system based on a foreign compound development toxicity molecular mechanism is established at a cellular level, and large-scale in-vitro high-throughput screening of the foreign compound is carried out, so that the defects of time consumption, labor consumption and the like of human or animal experiments can be avoided. Human umbilical cord mesenchymal stem cells (WJ-MSCs) are primitive stem cells with sub-totipotent differentiation potential existing in umbilical cord. At present, the embryonic stem cells are more suitable for in vitro screening of the developmental toxicity of exogenous compounds than other methods due to the characteristics of simple and convenient sources, high sensitivity to tested substances and the like.

Disclosure of Invention

The invention aims to provide a cell model for screening developmental toxic exogenous compounds by taking 11 beta-HSD 2 as a target spot, wherein cells are humanized, come from fetal umbilical cords, do not relate to complex ethical problems and are easy to obtain.

The second purpose of the invention is to provide a construction method of a cell model for screening developmental toxicity exogenous compounds by taking 11 beta-HSD 2 as a target spot, and the modeling method is simple and has strong repeatability.

The invention also aims to provide application of a cell model for screening developmental toxicity exogenous compounds by taking 11 beta-HSD 2 as a target point in detecting exogenous compounds with developmental toxicity, wherein the model has the advantages of rapid detection process and wide application range, and can be used for high-throughput screening.

One of the purposes of the invention adopts the following technical scheme:

a cell model for screening developmental toxic exogenous compounds by taking 11 beta-HSD 2 as a target point is obtained by adopting human WJ-MSCs as target cells and transfecting a luciferase reporter gene containing a 11 beta-HSD 2 promoter into the target cells.

The cell model for screening the developmental toxic exogenous compound by taking the 11 beta-HSD 2 as a target point has two indexes, namely 11 beta-HSD 2 gene expression and luciferase activity of a report system.

The second purpose of the invention is realized by adopting the following technical scheme:

a construction method of a cell model for screening developmental toxic exogenous compounds by taking 11 beta-HSD 2 as a target point comprises the following steps:

step 1: extracting human WJ-MSCs with 5% CO at 37 ℃ +/-1₂Culturing in an incubator and propagating for 3-5 generations;

step 2: constructing a luciferase reporter gene containing an 11 beta-HSD 2 promoter;

and step 3: and (3) transfecting the luciferase reporter gene containing the 11 beta-HSD 2 promoter constructed in the step (2) into the WJ-MSCs after passage in the step (1) to obtain the cell model for screening the developmental toxic exogenous compound based on the 11 beta-HSD 2 luciferase reporter gene.

The third technical scheme for realizing the purpose of the invention is as follows:

an application of a cell model for screening a developmental toxicity exogenous compound by taking 11 beta-HSD 2 as a target point in screening the exogenous compound with developmental toxicity.

When 11 beta-HSD 2 gene expression and luciferase activity of the reporter system are reduced, it is suggested that the exogenous compound to be screened has developmental toxicity.

The developmentally toxic exogenous compound comprises one or more of synthetic compounds, natural products, organic small molecules, inorganic small molecules, lipids and saccharides.

The developmentally toxic exogenous compounds include caffeine, nicotine, ethanol, and azithromycin.

Compared with the prior art, the invention has the following beneficial technical benefits:

1. compared with other in vitro screening systems for developmental toxic compounds, the cell model for screening the developmental toxic exogenous compounds by taking 11 beta-HSD 2 as a target point provided by the invention has the following advantages of used WJ-MSCs: compared with adult stem cells, the somatic stem cells are closer to human embryonic stem cells, can be induced and differentiated into almost all tissue cells in vitro, and can fully reflect the development process of tissues; secondly, the cells are from the umbilical cord, so that the obtained mother fetus is not damaged, and the problem of complicated ethics is not involved; the extraction and identification method is mature, and the obtained cells have high purity; fourthly, the growth environment is simple, and the reaction is sensitive to developmental toxic compounds; fifthly, expresses various genes participating in cell proliferation, tissue generation and organ development and can be used as a prediction index of the development toxicity of the exogenous compound.

2. In the construction method of the cell model for screening the developmental toxic exogenous compound by taking 11 beta-HSD 2 as the target point, the luciferase is an ideal reporter gene, and the requirements of specific target point, high sensitivity and high flux of the in vitro screening model are met. The mammal cell does not contain endogenous luciferase, the luciferase gene is inserted into a gene promoter to be analyzed through a molecular biology cloning technology and then is transfected into a cell chromosome to become a reporter gene expressed by the gene, the luciferase enables a substrate to emit fluorescence in the presence of oxygen, the binding specificity of the enzyme and the substrate is very strong, the detection sensitivity is high, the signal to noise ratio is higher due to the fact that non-specific interference of exciting light is avoided, and the advantages that other reporter genes cannot be replaced are achieved.

3. The cell model for screening the developmental toxicity exogenous compound by taking the 11 beta-HSD 2 as the target takes the luciferase activity as a detection index, and when the luciferase activity of the 11 beta-HSD 2 gene expression and report system is reduced, the exogenous compound to be screened is prompted to have developmental toxicity, is simple, high in specificity, high in sensitivity and wide in application range, can be used for high-throughput screening, and has important significance for rapidly screening the exogenous compound with developmental toxicity.

Drawings

FIG. 1 is a diagram showing the identification of human WJ-MSCs in example 1 of the present invention;

in the figure, A is the morphology of WJ-MSCs, and B is the expression of WJ-MSCs marker analyzed by flow cytometry.

FIG. 2 is a diagram showing the basic skeleton of a vector (GV238) as a tool for constructing a reporter gene system in example 1 of the present invention.

FIG. 3 is an electrophoresis diagram showing the cleavage result of the tool vector (GV238) used in the construction of the reporter gene system in example 1 of the present invention;

in the figure, 1 is 10kb Marker (the bands are from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp and 250bp in sequence), 2 is the vector enzyme digestion product, and 3 is the vector which is not digested.

FIG. 4 is an electrophoretogram of recombinant clones identified by PCR in example 1 of the present invention;

in the figure, 1 is a negative control (ddH2O), 2 is a negative control (no-load self-ligation control group), 3 is a positive control (GAPDH), 4 is a Marker (bands are sequentially 5Kb, 3Kb, 2Kb, 1.5Kb, 1Kb, 750bp, 500bp, 250bp and 100bp from top to bottom), and 5-12 is 11 beta-HSD 21-8 transformant.

FIG. 5 is a graph showing the detection of 11 β -HSD2 mRNA expression in WJ-MSCs after treatment with test compounds of example 1 according to the present invention;

in the figure, A is the expression of 11 β -HSD2 mRNA in WJ-MSCs treated with caffeine, B is the expression of 11 β -HSD2 mRNA in WJ-MSCs treated with ethanol, C is the expression of 11 β -HSD2 mRNA in WJ-MSCs treated with nicotine, and D is the expression of 11 β -HSD2 mRNA in WJ-MSCs treated with azithromycin (compared with the control,^*P<0.05，^**P<0.01)。

FIG. 6 is a graph showing luciferase activity in WJ-MSCs detecting transfection reporter systems after treatment with test compounds in example 1 of the present invention;

in the figure, A is the luciferase activity in WJ-MSCs under caffeine treatment, B is the luciferase activity in WJ-MSCs under ethanol treatment, C is the 11 beta-HSD 2 luciferase activity in WJ-MSCs under nicotine treatment, D is the 2 luciferase activity in WJ-MSCs under azithromycin treatment (compared with the control,^*P<0.05，^**P<0.01)。

figure 7 is a graph of the expression of fetal liver 11 beta-HSD 2 mRNA detected after azithromycin exposure during pregnancy in example 2 of the invention (compared to control,^*P<0.05，^**P<0.01)。

FIG. 8 is a graph showing the change in body weight and IUGR rate of fetal mice measured after azithromycin exposure during pregnancy in example 2 of the present invention;

in the figure, A is the weight of a fetal rat, B is the IUGR rate of a fetal rat (compared with the control group,^*P<0.05，^**P<0.01)。

FIG. 9 is a graph of indicators associated with liver development in fetal mice detected after exposure to azithromycin during pregnancy in example 2 of the present invention;

in the figure, a is fetal liver weight, B is fetal liver hematoxylin-eosin staining, C is fetal liver tissue differentiation development related gene mRNA expression (compared with the control group,^*P<0.05，^**P<0.01)。

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

The specific operation steps and procedures of the present invention will be further described in detail below with reference to examples.

Example 1 construction of cell model for screening developmental toxic foreign Compound with 11 β -HSD2 as target

1. WJ-MSCs acquisition

The research of human body specimens strictly conforms to the declaration of Helsinki of the world medical Association, the collection of umbilical cord tissues obtains the informed consent of parents of newborn and signs the informed consent of paper edition, and the experimental method passes the approval of related ethical committees. Taking umbilical cord of fetus born by caesarean section under aseptic condition in operating room, cleaning blood, soaking in pre-cooled normal saline, and returning to laboratory.

2. WJ-MSCs extraction

The umbilical cord is taken out from the superclean bench, and all separation processes are carried out in pre-cooled physiological saline. Cut into pieces of 4-6 cm/length, and washed with a syringe to remove blood. Removing umbilical cord adventitia, umbilical artery and umbilical vein, and stripping Wharton's gum: fixing two ends of umbilical cord on wood plate with tack, cutting the outer membrane from one end as inlet, separating the outer membrane while separating, separating the outer membrane from the two sides, clamping the outer membrane and Wharton's gum with two hemostatic forceps, and applying forceTearing, completely separating the outer membrane, and carefully separating the artery and vein. Wharton gum was torn into strips as much as possible and transferred to vials. Cutting with ophthalmic scissors to obtain meat paste, transferring into 50mL centrifuge tube, adding 40mL digestive juice (containing 40mL DMEM/F121: 1 culture medium and 80mg collagenase I), sealing with sealing membrane, mixing, digesting in 37 deg.C water bath for about 4 hr (or in 37 deg.C temperature controlled shaking table), and shaking once every half hour. After digestion is completed, 250mL of PBS is added for repeated blowing and dilution, 50mL of sterile centrifuge tubes are subpackaged, and cells are collected after centrifugation at 2500rpm for 15-20 min. Discarding the supernatant and the floating adipose tissues, adding about 5mL of DMEM/F12 complete medium containing 10% fetal calf serum and 1% streptococcal double antibody, and blowing and beating to prepare cell suspension; and the number of cells was counted under an inverted microscope using a cell counting plate. Placing at 37 ℃ and 5% CO₂And culturing in an incubator with 95% humidity.

3. Culture of WJ-MSCs

The obtained WJ-MSCs were mixed at 1X 10⁴Inoculation to 25cm at a density of/ml²Placing in a culture flask, standing in a constant temperature incubator at 37 deg.C for culturing, and maintaining CO²The concentration was 5%. Observing the adherence condition of the WJ-MSCs under an inverted microscope after 48 hours, wherein the cells with normal WJ-MSCs with good activity have regular shapes, most of the cells grow like fusiform fibroblasts, and the few cells are multi-protuberant astrocytes, have refractivity, large and irregular cell nucleuses and obvious nucleoli. If the WJ-MSCs adhere well and there are few or no obvious suspension cells in the upper medium, the medium can be changed immediately. If more cells were suspended in the upper medium, the complete culture medium was replaced with DMEM/F12 on day 3 after inoculation. The culture medium is replaced every other day, and the adherent growth condition of the cells is observed by using an inverted microscope. And (5) when the wall adherence of the WJ-MSCs reaches 80-90 percent of passage. During passage, firstly sucking the culture medium in the culture bottle, washing for 2 times by using 5mL of PBS buffer solution, adding about 1mL of 0.05% EDTA-Trypsin into the culture bottle, taking the bottle bottom as a limit, placing the culture bottle in an incubator for digestion for 2-3 min, observing the WJ-MSCs under an inverted microscope to cause the gap to grow and the cells to become round and bright, namely dripping 5mL of complete culture medium (DMEM/F12 culture medium containing 10% FBS) into the culture bottle to stop digestion, and slightly beating the side of the culture bottle for several times. DMEM/F12 complete medium was added again in an amount of 5mL, andrepeatedly and lightly blowing the bottom of the bottle by a pipettor to ensure that all the WJ-MSCs fall off from the bottom wall of the culture bottle, and evenly dividing the WJ-MSCs suspension into another 2 new 25cm²Placing in a cell culture bottle, placing in a constant temperature incubator at 37 deg.C, maintaining CO²The culture was carried out at a concentration of 5%.

4. Flow cytometry identification of WJ-MSCs

Expanding the extracted WJ-MSCs to 3 rd generation, pouring out the culture medium, and adding 5m of culture medium; and (3) lightly blowing PBS for washing for 1 time, removing PBS, adding about 1mL of 0.05% EDTA-Trypsin respectively, placing the mixture in an incubator for digestion for 2-3 min, inverting the microscope to observe that gaps among the WJ-MSCs become larger and cells become round and bright, namely dropping 5mL of a complete culture medium (DMEM/F12 culture medium containing 10% FBS) into the culture bottle respectively to terminate digestion, lightly beating the side of the culture bottle for a plurality of times, and lightly blowing bottom cells of the bottle repeatedly to ensure that all the WJ-MSCs fall off from the bottom wall of the culture bottle. Collecting cells into a 15mL centrifuge tube, centrifuging at 1500rpm × 10min with a horizontal centrifuge, discarding the upper layer culture medium, adding 5mL PBS, blowing, resuspending, washing for 1 time, and centrifuging at 1500rpm × 10min with a horizontal centrifuge. Discard PBS supernatant, leave about 100. mu.L, wrap with tinfoil, and operate away from light. 5 μ L of FITC labeled CD29, CD90, CD34, CD45, Isotype IgG1 antibody were added, and the cells were blown and resuspended several times. The resuspended cells were incubated in a refrigerator at 4 ℃ for 30min, then 5mL PBS was added, blown, resuspended, and washed 1 time. The mixture was centrifuged at 1500rpm X10 min in a horizontal centrifuge. Discarding the supernatant, adding PBS 400 μ L, blowing 1mL suction head for resuspension, preparing cell suspension 0.5mL again, adjusting the concentration to 1X 10⁶the/mL is sent for immediate testing and filtered into a dedicated assay tube with a dedicated filter before testing.

5. Construction of recombinant vector for reporter Gene System

The 11 beta-HSD 2 promoter fragment required for cloning from genome DNA by using a PCR method, and the 11 beta-HSD 2 promoter gene sequence is shown as SEQ ID NO.19, and the fragment is inserted into a tool vector (GV238) containing a luciferase reporter gene. And transfecting the constructed recombinant vector into a competent cell, carrying out colony PCR identification on the grown clone, and then carrying out sequencing and comparison analysis on the clone with positive PCR identification, wherein the result that the comparison is correct is that the construction of the recombinant vector of the reporter gene system is successful.

6. Transfection of cells of interest

Preparing cell suspension from WJ-MSCs in logarithmic growth phase, counting, and inoculating to 24-well culture plate (cell number is about 2 × 10)⁴Specifically according to the size of the cell morphology), 37 ℃ and 5% CO₂The incubator is incubated until the degree of cell confluence reaches about 80%. According to the cell transfection preliminary experiment and the design of an invitrogen lipofectamine 2000 transfection reagent using instruction, appropriate amount of successfully constructed reporter gene system recombinant vector and transfection reagent are added. After 4-6h, the cell status was observed and replaced with fresh complete medium. Observing the expression condition of the fluorescence labeling gene on the plasmid after 24-48h of transfection, wherein the fluorescence rate is more than 80%, supplementing 500 mu L of normal culture medium after the fluorescence is beaten, and waiting for further treatment.

7. WJ-MSCs administration

According to the literature, 4 developmentally toxic exogenous test compounds are selected, and the dissolved test compounds are: caffeine (0, 0.1, 1.0, 10, 100. mu.M), nicotine (0, 0.1, 1, 10, 100. mu.M), ethanol (0, 15, 30, 60mM), azithromycin (0, 1.6, 8, 40. mu.M) were added to the cultured cells in a concentration gradient and treated for 24 h. After the treatment is finished, the cell culture medium of each well is discarded, the cells are washed by PBS, and then 200 muL of Trizol is added into each well for extracting the total RNA of the cells, or 200-500 muL of cell lysate is added for detecting the luciferase activity.

8. Specific primer design and preparation

Designing a primer: primers were designed and verified using Primer Premier 5.0 and NCBI Blast database. After designing the primer sequence, it was synthesized by Biotechnology engineering (Shanghai) GmbH and purified by PAGE. Centrifuging 7500g of EP tube filled with newly synthesized primers for 10 minutes, adding molecular biological ultrapure water with the volume marked on the tube wall, and shaking and mixing uniformly for later use.

TABLE 1 RT-PCR primer sequences

9. Detection of luciferase Activity of WJ-MSCs

9.1 initial use

When the Reporter Assay System is used, the Luciferase Assay Buffer II needs to be dissolved and balanced at room temperature in advance; completely adding Luciferase Assay Buffer II into a Luciferase Assay Substrate bottle, completely dissolving a Substrate to form Luciferase Assay Reagent, subpackaging and storing at-80 ℃, and being effective within one year.

9.2 before cell Lysis, 5X Passive lysine Buffer was diluted with D-Hanks to 1X; absorbing the culture medium in the 24-well plate, adding 300 μ L of Passive Lysis Buffer 1 ×, placing in a refrigerator at 4 deg.C for 20min to allow the cells to fully lyse, shaking for 3-5min (not too violent), mixing well, and immediately detecting, or placing in a refrigerator at-80 deg.C for one week to allow detection.

9.3 before the computer is operated to detect, Stop is taken in advance&

Buffer is placed at room temperature for dissolution and equilibration, Stop is obtained&

Substrate 50X addition to Stop&

In Buffer, the mixture was dissolved sufficiently and diluted to 1 × Reagent. Stop&

The Substrate 1 × Reagent needs to be prepared for use, and is effective within 48 hours at normal temperature after being prepared.

9.4 dissolving the cell lysate in the step 9.2 at normal temperature, sucking 40 mul into a Lockwell maxisorp detection plate, adding 20 mul of Luciferase Assay Reagent, shaking and mixing uniformly, immediately detecting firefly luminescence by using a microplate reader, and paying attention to the step that the time is not longer than 5 min.

9.5 after detection of firefly luminescence, 20. mu.L of Stop was added to each well&

Reagent, shaking, mixing uniformly, standing for 3min, and detecting Renilla luminescences (fluorescence value of Renilla luciferase) by using an enzyme-labeling instrument.

9.6 data collection and analysis.

SPSS 17 was used for data analysis. Metrology data is expressed as Mean ± standard error (Mean ± SEM). The Student's t-Test pair is used for testing the difference between the mean values of the two samples; comparisons between the mean of multiple samples were performed using one-way ANOVA analysis of variance. P <0.05, indicating that the difference was statistically significant.

10. Results of the experiment

10.1 WJ-MSCs identification

The WJ-MSCs were passaged to the third generation and the cell morphology was observed with an inverted microscope. The cells are adherent, uniform in shape, parallel in arrangement or vortex in growth, part of the cells are in a fibroblastic shape, the cells have refractivity, the cell nucleus is large and irregular, and the nucleolus is obvious (figure 1A). Flow cytometry identification of cell surface antigen molecules is carried out, and the WJ-MSCs are found to strongly express mesenchymal stem cell surface markers CD29 and CD90, and the positive cell rate is 100%. No hematopoietic stem cell surface marker CD34 and no leukocyte surface marker CD45 were expressed and positive cells expressed less than 2% (fig. 1B). The result indicates that the extracted WJ-MSCs have typical characteristics of mesenchymal stem cells and meet the international identification standard of the mesenchymal stem cells.

10.211 beta-HSD 2 luciferase reporter gene system vector construction

The GV238 vector used was digested and electrophoresed, and it was found that the digested product was strongly positively expressed at 5kb (FIG. 3). The electrophoresis result of the PCR product shows that the PCR product of the 11 beta-HSD 2 positive transformant is between 750bp and 1Kb (figure 4), and meanwhile, the sequencing result shows that the sequence is 100 percent consistent with the GenBank alignment. The result shows that the product of the recombinant clone is 11 beta-HSD 2, and the constructed recombinant vector is a luciferase reporter gene system vector containing 11 beta-HSD 2 promoter.

10.3 Effect of test Compound treatment on WJ-MSCs 11 β -HSD2 mRNA expression

Caffeine, nicotine, ethanol, and azithromycin all decreased concentration-dependently in WJ-MSCs the expression of 11 β -HSD2 mRNA (P <0.05 or P <0.01, fig. 5A-D) compared to the control group.

10.4 Effect of test Compound treatment on luciferase Activity of WJ-MSCs

The luciferase activity in WJ-MSCs transfected with the 11 β -HSD2 promoter luciferase reporter gene was reduced in a concentration dependent manner (P <0.05 or P <0.01, FIGS. 6A-D) following caffeine, nicotine, ethanol and azithromycin treatment, as compared to the control group.

[ example 2 ] developmental toxicity verification of exogenous developmental toxic Compound Azithromycin based on screening in example 1

1. Laboratory animal

SPF grade healthy Kunming mice are purchased from the disease prevention and control center of Hubei province, and the animal license number is as follows: SCXK (jaw), No.2017 and 0018. The study was approved by the ethics committee of the department of medicine of wuhan university and was performed strictly in accordance with the relevant treatment guidelines of the international laboratory animal protection certification and evaluation agency.

2. Animal treatment

Healthy adult Kunming mice were purchased from the disease control center in Hubei province, with female weights of 25 + -5 g and male weights of 30 + -5 g. The females and males were combined at 2:1 every 6 nights, and the following morning the vaginal and sperm smears were examined to determine whether the females had successfully received the pregnancy and scored as pregnancy day 0 (GD 0). Pregnant rats were randomized into three groups: control group (Control), low-dose azithromycin exposure group (AL), and high-dose azithromycin exposure group (AH). At 8-10 am each day starting at GD9, the AL and AH groups were given azithromycin (37.5 or 150mg/kg. d), respectively, and the control group was given an equal volume of saline. 2h after GD18 last administration, pregnant mice were anesthetized with isoflurane and then laparotomized to obtain fetal mice (n is 10), and the weight of the fetal mice was recorded, and the average value of the weight of the control group fetal mice minus 2 standard deviations was used as the judgment standard of intrauterine growth retardation (IUGR) fetal mice. Fetal liver tissue was then weighed, snap frozen in liquid nitrogen and stored at-80 ℃. Optionally, 5 livers per group were fixed in 4% paraformaldehyde and HE stained.

3. Fetal liver morphology detection

The fetal liver tissues are taken and fixed for 48 hours, dehydrated, soaked in wax, embedded and sliced, dewaxed to water, stained with hematoxylin-eosin, observed under a microscope and photographed.

4. Fetal liver gene expression detection

Extracting total RNA of liver tissue of mice in the inner tube of the uterus, carrying out reverse transcription to obtain cDNA, and carrying out quantitative analysis on the expression condition of related genes by RT-PCR. The primer sequences of the genes are shown in Table 2:

TABLE 2 RT-PCR primer sequences

5. Results of the experiment

5.1 Effect of azithromycin exposure during pregnancy on the expression of 11 beta-HSD 2 in fetal rat liver

As shown in figure 7, 11 β -HSD2 expression was reduced in AL and AH groups after azithromycin exposure during pregnancy compared to the control group (P <0.01, figure 7).

5.2 Effect of Azithromycin Exposure on fetal rat body weight and IUGR Rate during pregnancy

As shown in fig. 8, fetal mice in AL and AH groups lost weight (P <0.05 or P <0.01, fig. 8A) and IUGR rates increased (P <0.01, fig. 8B) after azithromycin exposure during pregnancy compared to the control group. It is suggested that azithromycin exposure during pregnancy may affect the overall development of fetal rats.

5.3 Effect of Azithromycin Exposure on fetal rat liver development during pregnancy

As shown in fig. 9, the weight of fetal liver in AH group was reduced after azithromycin exposure during pregnancy (P <0.01, fig. 9A), and vacuolization of hepatocyte cytoplasm in AL group and AH group was evident compared to control group (fig. 9B). The mRNA expression of the developmental genes ALB, PCNA and HNF4 alpha of the fetal liver is reduced, and the mRNA expression of the developmental genes AFP and caspase-3 is inhibited to be increased (P <0.05 or P <0.01, figure 9C). It is suggested that azithromycin exposure during pregnancy may cause fetal liver dysplasia.

In conclusion, the test compound to be screened is given different concentrations on a WJ-MSCs proliferation cell model, the intracellular 11 beta-HSD 2 mRNA expression and the intracellular luciferase activity of the 11 beta-HSD 2 promoter luciferase reporter gene are detected, and if the test compound to be screened can reduce the 11 beta-HSD 2 mRNA expression and the luciferase activity, the test compound to be screened is suggested to have developmental toxicity. Then, the invention further exposes the tested compound screened out by the female mouse in the pregnancy period on the whole animal model, proves that the adverse effect on the whole development and the organ development of the fetal mouse is consistent with the result of the cell model which is constructed by the invention and screens out the developmental toxicity exogenous compound by taking 11 beta-HSD 2 as a target point. The exogenous compound screening system established by the invention has the characteristics of high specificity, high sensitivity, high stability, high feasibility and the like, can be used for screening high-flux exogenous compounds, and has important significance for detecting exogenous compounds with developmental toxicity.

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gcctgagagt ccatacttgt tag 23

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

actccactca cggcaaattc 20

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccagtagact ccacgacata ct 22

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aactgcgtga cctctgttc 19

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cacgtcagcc actacgatat t 21

<210> 19

<211> 2116

<212> DNA

<213> 11β-HSD2(11β-HSD2)

<400> 19

cttcaactcc tgctttagca agtgctggtt ctatctgtgt tgccttgacc ttgaaggctc 60

cccaggaccc tcatctcagc ttctactccc ttcctcactc caaggggaag actccaatgg 120

actgaggctg gagaaaccac agccagactc ttggtgtggt ggctaaagct ggggggtctg 180

gccaatccaa tctctacagc tcaaatcagg aagtcaggcc aggggtgctg tgtctgcctc 240

caagcctggg ccatacattc accgcaacag gaatcagtga ctttggaaaa ggaatgtttc 300

cctgagtttc tagaggcagg atgacccaga ccgaaggaca gagagttacc agaaacacac 360

acaatgcaca caaatgtact cacttgcaca cacagggtca tggcataggg agtgacacaa 420

ccaaatggcc tggacaagcc acagagtccc atggacacaa gtgcttacaa acacaggcac 480

acaaggtcat gtacccaagg tcaggcacac acaccctcct cattcaagtc ctgcctccca 540

ggtctggggc ttgcctgtca tctgtcctta ggaggaggga taggtggagg cacaggagtg 600

gctcagggca aattggtcct ccacccagag actctccctt taggtgggtc tggctcccag 660

cctcccctga gatttccctg gctcaccagg gggctgtgtc tggtggcaga gagggctttg 720

gaggaggcat tgccctgaaa ccctggcaga gcctgggcac ctaagagaga atgagcaaca 780

gggctaggaa gtgtgacagg gagtccacaa cctcccagga tcttgggagg gcagggatga 840

ggcactgagc acggcctgga agcccacggg aagcagggac ccctctgtcg agggctacac 900

ctactgctct cagcatcaag cctgcagcca ggtactggca ccaactccca gtgcctggca 960

gagccctggc atggatgcta ggcacagggt gccagacacg ggggtgggca gggtgggggg 1020

ccactgggcc acacacaagc atggagacct tcacacacgc aggcagaaaa atccacccac 1080

actcatacca cttattttac cccctggggg ttcaggggag caaggcaggg accaccctgt 1140

cctagaggct gccttcaccg acgtccctct ggcctggctc ttggggttgg cgggggtggg 1200

ccataagtaa tgggagacct aggtgcttcc tctgacaccc caccctccac tgacccccac 1260

acacatacat ttgtgaggca ggaagtgggg ttgtgcgtcc ccaggtttaa gccctggaag 1320

gaaagggaaa gacggggcag agactccaag aaccgggagc ggtcgtcctg ttcccccgcc 1380

agcccctgtc ccaggcaggt tttgtggccg cgagctgctc aggggtgagc gcgccttagg 1440

gtgtgtgcgc cagggacctg gcgcagccga ccacgtggga gtgcggcgcg tgtacgggcg 1500

cgggcggcag cggcagcggc agcggagacc gggtgagcac cggctggttc ctcgcggtgt 1560

tcctgcaggc ttggcggccg cggtggtgcg atcagcaaag ggcaccggga tgccggttgt 1620

gcgtgtcctc aggtgtcccg aacaagcgtg agtggcatgt gctcacctga gcgcggcggc 1680

ttggcggccc cgggcgtgtg ggtgggggcg cggcggagag ctgaggccgg gcacccgggc 1740

tggaccgacg ggccctctcc caagcaccgc ccgcagccag gcggctcctc gagcgcagca 1800

actttgggac tttgttccgg ctttttccaa atcgaatctg gtcgaggggg cggggcgggg 1860

ggggagcacc tgccctgcgg ggggtgccgg gagagaagtg aaggaaagcc ccgcccccgc 1920

ccctcccgct ccccgccctc tcccccgccc ccggggctct tcataagctc ggcccgaggg 1980

cgagcagaga aagcgagtgt ccctctcgcg ccccaggccg gtgtaccccc gcactccgcg 2040

ccccggccta gaagctctct ctccccgctc cccggcccgg cccccgcccc gccccgcccc 2100

agcccgctgg gccgcc 2116

Claims

1. An application of a cell model for screening a developmentally toxic exogenous compound by taking 11 beta-HSD 2 as a target point in screening the developmentally toxic exogenous compound with abnormal fetal liver development is characterized in that: comprises the following steps:

and step 3: transfecting the luciferase reporter gene containing the 11 beta-HSD 2 promoter constructed in the step 2 into the WJ-MSCs after passage in the step 1 to obtain a cell model for screening development toxicity exogenous compounds based on the 11 beta-HSD 2 luciferase reporter gene; the developmental toxicity exogenous compound is azithromycin, and the abnormal development of fetal liver caused by the exposure of azithromycin in the pregnancy period is shown as follows: the mRNA expression of the development promoting genes ALB, PCNA and HNF4 alpha of the fetal liver is reduced, and the mRNA expression of the development inhibiting genes AFP and caspase-3 is increased.