CN114703277B

CN114703277B - Application of ADORA2A gene as molecular marker in preparation of product for preventing or relieving IUGR pregnancy outcome

Info

Publication number: CN114703277B
Application number: CN202210462177.8A
Authority: CN
Inventors: 谭成全; 张玲娜; 伍子放; 崔志娟; 徐铮; 冯莉; 印遇龙
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-04-28
Filing date: 2022-04-28
Publication date: 2022-11-01
Anticipated expiration: 2042-04-28
Also published as: CN114703277A

Abstract

The invention discloses an application of ADORA2A gene as a molecular marker in preparing a product for preventing or relieving IUGR pregnancy outcome, belonging to the technical field of medicines. According to the invention, an in vivo test shows that the Adenosine/ADORA2A signal can be used as a molecular marker for relieving the IUGR pregnancy ending of the female mouse, a powerful theoretical basis and technical support are provided for relieving the IUGR pregnancy ending of the female mouse, the defects and the defects of the traditional detection technology are overcome, and the kit has the advantages of higher safety, better effect, lower cost, higher accuracy, sensitivity and specificity and the like, and provides revelation and possibility for the subsequent research and preparation of products for preventing and treating the IUGR pregnancy ending.

Description

Application of ADORA2A gene as molecular marker in preparation of product for preventing or relieving IUGR pregnancy outcome

Technical Field

The invention relates to the technical field of medicines, in particular to application of ADORA2A gene as a molecular marker in preparation of a product for preventing or relieving IUGR pregnancy outcome.

Background

Intrauterine growth restriction (IUGR) is the most common form of embryonic dysplasia, manifested by failure of the fetus to reach the size due to its genetic factors. IUGR not only leads to increased neonatal mortality and morbidity, but also has a permanent negative impact on the future growth of the neonate. Among the factors affecting the occurrence of IUGR, the nutrition supply from the mother to the fetus is the most direct and main factor, during the whole pregnancy, the nutrients required by the fetus are all from the mother, and the nutrients in the mother are transported to the endometrium along with the blood, then collected to the umbilical cord through the placenta and transported to the fetus. Therefore, the generation state of placenta angiogenesis in gestation period is closely related to the occurrence of IUGR.

Adenosine, an endogenous nucleoside, regulates various physiological processes under normal and pathological conditions by activating four different G-protein coupled adenosine receptors. Adenosine has a direct mitogenic effect on vascular endothelial cells, and its ability to modulate vascular endothelial cells and immune cells to produce pro-and anti-angiogenic substances, suggesting the potential of adenosine for angiogenic biological events. In past studies, although studies have indicated that adenosine signaling may play a critical role in embryonic development, there has been no report on the regulation of adenosine on placental angiogenesis during pregnancy and its association with IUGR.

Disclosure of Invention

The invention aims to provide application of ADORA2A gene as a molecular marker in preparing a product for preventing or relieving IUGR pregnancy fate, so as to solve the problems in the prior art, verify that the IUGR pregnancy fate is related to poor placental angiogenesis through experiments, and improve adenosine metabolic level and adenosine receptor (ADORA 2A) expression by supplementing adenosine, thereby promoting placental angiogenesis and preventing or relieving IUGR pregnancy fate.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides application of ADORA2A gene as a molecular marker in preparing a product for preventing or relieving IUGR pregnancy outcome.

Preferably, the product for preventing or alleviating the pregnancy outcome of IUGR comprises a product for detecting the expression level of the ADORA2A gene in a fetal placental tissue to be tested, and the expression level of the ADORA2A gene being lower than the expression level in a normal developing fetal placental tissue indicates that the fetus to be tested is developing IUGR.

Preferably, the ADORA2A gene is administered to prevent or alleviate the occurrence of IUGR pregnancy outcome by promoting fetal placental angiogenesis.

Preferably, adenosine metabolism in the fetal placenta can be improved and the expression of the ADORA2A gene can be re-up-regulated by supplementing adenosine, so as to promote fetal placental angiogenesis, and prevent or relieve the occurrence of IUGR pregnancy fate.

Preferably, the IUGR pregnancy outcome comprises: at least one of a decrease in fetal weight and fetal length, a decrease in placental efficiency, an increase in embryo absorption rate, an increase in IUGR rate, a decrease in the area of the maze area of the placenta, and a decrease in the proportion of sinusoid area.

Preferably, the product for preventing or alleviating the pregnancy outcome of IUGR comprises: the products of IUGR pregnancy outcome are detected by PCR, RT-PCR, real-time quantitative PCR, DNA sequencing, immunodetection, protein chip, in situ hybridization or gene chip which take genome DNA in the placenta tissue of the fetus to be detected as a template.

Preferably, the product for detecting IUGR pregnancy outcome comprises at least one pair of primers for specifically amplifying the ADORA2A gene by PCR, RT-PCR, real-time quantitative PCR and DNA sequencing.

Preferably, the primers comprise ADORA2A-F and ADORA2A-R, and the nucleotide sequences are as follows:

ADORA2A-F(SEQ ID NO:1)：5’-AGCAACCTGCAGAACGTCACAAAC-3’；

ADORA2A-R(SEQ ID NO:2)：5’-TGGCAATAGCCAAGAGGCTGAAGA-3’。

preferably, the product for detecting IUGR pregnancy outcome by the immunoassay and protein chip comprises: antibodies, including polyclonal and monoclonal antibodies, that specifically bind to the protein encoded by the ADORA2A gene.

Preferably, the product for detecting IUGR pregnancy outcome by said in situ hybridization or gene chip comprises: a probe that hybridizes to a nucleic acid sequence of the ADORA2A gene.

The invention discloses the following technical effects:

(1) The invention discovers that serious poor angiogenesis conditions exist in IUGR placenta by comparing the angiogenesis and adenosine expression levels of normal and IUGR fetal placenta tissues of different species, and the poor angiogenesis conditions comprise the down regulation of the levels of angiogenesis-related factor protein and mRNA; with a concomitant decrease in ADORA2A levels.

(2) The invention discovers for the first time that the occurrence of IUGR of pregnant female rats can be relieved by supplementing 0.1 percent of adenosine to drinking water. Provides a powerful reference basis for preventing the pregnancy fate of the IUGR of the female rat, directly targets the biological event of the placental angiogenesis compared with the previous methods starting from the aspects of formula or feed raw materials and the like, has more ideal improvement effect and more scientific improvement mechanism.

(3) The invention has important guiding significance for preparing related products for preventing and treating IUGR, is expected to provide important theoretical basis and technical support for developing nutrition strategies or products for preventing and treating IUGR in pregnancy, overcomes the defects and shortcomings of the traditional detection technology, has the advantages of higher safety, better effect, lower cost and the like, and provides revelation and possibility for preparing products for preventing and treating IUGR pregnancy outcome in subsequent research.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a comparison of angiogenesis and adenosine receptor expression in normal and IUGR fetal placentas from different species; wherein, A-B: mRNA levels of the different neonatal heavy mouse placental angiogenic factor (a) and adenosine receptor (B) genes; C-D: mRNA levels of different neonatal heavy piglet placental angiogenic factor (C) and adenosine receptor (D) genes; E-G: the levels of angiogenesis factors and adenosine receptors in different birth weight mice and piglet placentas.

FIG. 2 is a graph showing the effect of the addition of 0.1% adenosine to the drinking water on the IUGR of the pregnant mouse placenta; wherein, A: constructing an IUGR high-incidence model (IUGR group) and a normal group by using 6% of low-protein daily ration in the gestation period; B-C: the cumulative feed intake (B) and water intake (C) of pregnant mice in the gestation period; d: fetal placenta morphology of pregnant mice; E-Q: pregnant mice are 0.5 day heavy (E) in gestation, 18.5 day heavy (F) in gestation, weight gain in gestation (G), uterine embryo weight (H), number born (I), fetal weight (J), placental weight (K), fetal body length (L), placental diameter (M), placental efficiency (N), number of absorbed fetuses (O), embryo absorption rate (P) and IUGR incidence (Q).

FIG. 3 is a graph showing the effect of the addition of 0.1% adenosine to the drinking water on the placental angiogenesis in pregnant mice; wherein, A-H: the targeted metabolome detects changes in the content of metabolites of the adenosine metabolic pathway in the placenta, including adenosine (B), adenine (C), AMP (D), inosine (E), hypoxanthine (F), xanthine (G) and uric acid (H); I-N: PAS staining detects placenta maze area (bar =1 mm) (I) and HE staining detects placenta sinus area (bar =250 μ M in the first row, bar =50 μ M in the second row) (J), including placenta maze area (K), junction area (L), maze area/total placenta area (M), and placenta sinus area (N);

FIG. 4 is a graph of the effect of drinking water with 0.1% adenosine on the expression of placental adenosine receptors in pregnant mice; wherein, A-E: placenta angiogenesis related factor mRNA level (A) and protein expression (B-E); F-J: placental adenosine receptor mRNA expression (F) and protein expression (G-J); K-M: placental ADORA2A immunohistochemical staining, (first row bar =125 μm, second row bar =25 μm); N-O: placental ADORA2A was immunofluorescent stained with CD31 (bar =20 μm);

FIG. 5 is a graph of the effect of exogenous adenosine on pig vascular endothelial cell vascularization; wherein, A: effect of different concentrations of exogenous adenosine on endothelial cell viability (n = 6); B-C: effect of different concentrations of exogenous adenosine on endothelial cell migration capacity (bar =20 μm); D-E: effect of different concentrations of exogenous adenosine on endothelial cell tubulogenesis (bar =100 μm); f: effect of 100 μ M adenosine treatment on cellular cAMP levels

FIG. 6 effect of exogenous adenosine on cellular adenosine receptor expression. Wherein: A-D: effect of 100 μ M adenosine treatment on cell migration (bar =20 μ M) and tubulogenesis (bar =100 μ M) capacity; E-F: effect of 100 μ M adenosine treatment on cellular vascular associated factor and adenosine receptor mRNA levels; G-I: effect of 100 μ M adenosine treatment on cellular VEGF-Sub>A and adenosine Sub>A 2 Sub>A receptor protein expression;

FIG. 7 is a graph of the effect of drug regulation of ADORA2A on cell tubulogenesis; wherein, A-B: migration (bar =20 μm) and tubulogenesis (bar =100 μm) status following treatment of cells with nonselective adenosine receptor activator NECA and ADORA2A specific activator CGS 21680; c: cell viability; d: the distance of cell migration; e: the number of cell lumens;

FIG. 8 is a graph of the effect of silencing ADORA2A on cell tubulation; wherein, A-D: effect on cell migration (bar =20 μm) and tube forming ability (bar =100 μm) after siRNA transfection; e: effect on cellular angiogenic factor mRNA levels after siRNA transfection; f to J: detecting the protein levels of ADORA2A, ang, p-Akt and p-Stat3 in the cells after siRNA transfection; K-L: effect on cell tubulogenesis ability after siRNA transfection after treatment of cells with Akt signaling pathway activator Recilisib and Stat3 signaling pathway activator collividin, respectively (bar =100 μm);

FIG. 9 is a graph of the effect of overexpression of ADORA2A on cell tubulation; wherein, A-D: effect of lentivirus on cell migration (bar =20 μm) and tubulogenic (bar =100 μm) capacity following overexpression of adenosine A2a receptor; e: effect on cellular angiogenic factor mRNA levels after lentivirus overexpression; F-J: after lentivirus overexpression, the protein levels of ADORA2A, ang, p-Akt and p-Stat3 in cells were determined.

FIG. 10 shows that Adenosine/ADORA2A promotes the expression of Ang to promote cell tubulation by activating Stat3 and Akt signaling pathways; wherein, A-D: effect on cell migration (bar =20 μm) and tubulogenesis capacity (bar =100 μm) after overexpression of Ang; E-I: detecting the protein levels of ADORA2A, ang, p-Akt and p-Stat3 in the cells after the Ang is over-expressed; J-K: effect of overexpression of Ang on cell tubulogenesis ability following silencing ADORA2A (bar =100 μm); m to N: effect of overexpression of Ang on cell tubulogenesis after treatment of cells with the Akt signaling pathway inhibitors Artemisinin and Stat3 signaling pathway inhibitor Stattic (bar =100 μm); O-P: ang protein levels of cells after Artemisinin and Static treatment under adenosine treatment.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The principle of the invention is as follows:

according to the invention, C57BL/6J female mice are taken as research objects, and the fact that the farrowing performance of the female mice is remarkably reduced after the low-protein daily ration is fed by the low-protein daily ration with the mass fraction of 6% in the gestation period is found, wherein the performance comprises the reduction of fetal weight, the reduction of fetal body length, the reduction of placental weight, the reduction of placental efficiency, the increase of embryo absorptivity and the increase of IUGR rate, and the successful construction of an IUGR model is marked. Meanwhile, the IUGR placenta is found to have the reduction of the ratio of the area of a maze area of the placenta to the area of a blood sinus, and has a serious poor angiogenesis state; and is accompanied by impaired adenosine metabolism and decreased ADORA2A expression.

On the basis of the successful construction of the IUGR model, the drinking water is adopted to supplement adenosine with the mass fraction of 0.1%, and the following results are found: the supplementation of the drinking water with 0.1% adenosine significantly improved the development of IUGR fetal mice, including increasing fetal weight and fetal length, increasing placental efficiency, decreasing embryo absorption rate and IUGR rate, and improved the poor state of angiogenesis in the placenta, and increased the expression of placental ADORA2A, indicating that the supplementation of adenosine alleviated the occurrence of IUGR.

Furthermore, a porcine vascular endothelial cell construct external model is adopted, a gene expression regulation and control technology is adopted, the mechanism of promoting angiogenesis by Adenosine/ADORA2A signals is explored, and the mechanism is found to be possibly realized by promoting the expression of downstream Stat3, akt signal channels and Ang.

To further illustrate the above experimental principles, further details will be given by way of examples.

Example 1 comparison of the differential levels of angiogenesis and adenosine receptor expression in placental tissues of different species

1. Sample collection

Mouse placenta is collected from C57BL/6J pregnant female mice, and the female mice are divided into two groups according to the weight consistency principle after being successfully bred: the control group was fed with a growth and reproduction ration (carbohydrate content: 63.95%, protein content: 20.3%, fat content: 7%); the IUGR model group was fed an isocaloric low protein diet (carbohydrate content: 77.95%, protein content: 6%, fat content: 7%). The feeding period is 18 days of gestation, the female mouse is killed by cervical dislocation 18 days later, and placenta is collected for subsequent test.

The pig placenta samples were collected from long white x large white sows with good health and normal pregnancy. Feeding normal pregnancy daily ration in the gestation period, arranging testers to watch on the day of delivery, and immediately preparing consumables required by delivery after the sow is found to deliver. After the piglets are discharged, the umbilical cord close to the placenta is quickly tied up by a thin cotton thread (disinfected) with a digital label, and then the umbilical cord is cut at the position close to the fetus, so that the umbilical cord with the digital label is retracted to the birth canal. After the mucus on the piglets is wiped dry by a dry towel, the weights of the piglets are immediately weighed, and ear tags are marked on the ears of the piglets, so that the numbers of the ear tags of the piglets are consistent with the numbers of the digital tags on the umbilical cords, and the placenta can correspond to the ear tags of the piglets after being discharged. After the placenta is discharged, the individual placenta is quickly peeled off, and the excess umbilical cord is cut off. Stripping a connecting membrane between a placental membrane and a placenta at a position 3-4 cm below the root of the umbilical cord, collecting 3 placenta samples, quickly putting into liquid nitrogen, and subsequently storing at-80 ℃. One normal weight piglet placenta and one IUGR piglet placenta sample were collected from each sow. IUGR piglets were assessed as two standard deviations below the average primary weight of the control group.

Collecting and storing placentSub>A molecule sample, extracting RNA and reverse transcribing to obtain cDNA, and performing q-PCR test according to conventional method to detect the expression difference between angiogenesis related gene (VEGF-A, EGF, ang, HIF-1 alphSub>A, bFGF, TGF-1 betSub>A, PDGF-A) and adenosine receptor related gene (ADORA 1, ADORA2A, ADORA2B, ADORA) in different species of normal and IUGR placentSub>A. Wherein, the amplification Primer is designed by Primer 6, the sequence of the Primer is shown in table 1, the Primer is verified by Blast in NCBI, then synthesized by Guangzhou synthesis department of bioengineering biology engineering Co., ltd, and then fluorescence quantitative PCR test is carried out after the optimum annealing temperature is found out by ordinary PCR and the product unicity is verified. Using 384-well quantitative PCR plates, the reaction system was: 2. Mu.L of cDNA, 5. Mu.L of 2 XColor SYBR Green qPCR Master Mix, 0.2. Mu.L of upstream and downstream primers, 2.6. Mu.L of sterile H₂And O. The reaction procedure is as follows: 5min at 95 ℃;95 ℃ for 10sec,60 ℃ for 30sec, for 40 cycles. According to the threshold cycle (Ct) of the target gene and the internal reference, 2 is adopted^-ΔΔCtThe method calculates the relative expression level of mRNA of each target gene in a sample, and adopts 18S rRNA as an internal reference gene.

TABLE 1 Gene primer sequences

The collected and preserved placenta molecule sample is carried out with Western Blot test to detect the expression levels of four adenosine receptors and Ang and VCAM1 proteins. Ang (ab 95389), ADORA1 (ab 82477), ADORA2A (ab 3461), ADORA2B (ab 222901), ADORA3 (ab 197350), VCAM1 (ab 134047) antibodies were purchased from abcam corporation (USA); beta-actin (4970) and Anti-rabbitIgG (7074) antibodies were purchased from Cell Signaling Technology (CST) Inc. (USA).

2. Test results

The results are shown in fig. 1, and show that the gene level of the mRNA expression of IUGR fetal mouse placenta VEGF-A, ang and ADORA2A is significantly lower than that of a normal fetus (fig. 1A-B); the levels of VEGF-A, ang, bFGF, TGF-1 β, ADORA2A and ADORA2B mRNA in IUGR fetal pig placentSub>A are significantly lower than in normal fetuses (FIGS. 1C-D). The protein levels also showed that the protein expression levels of Ang, VCAM1 and ADORA2A in IUGR fetal mouse and fetal pig placenta were significantly lower than normal fetuses (fig. 1E-G). The results show that: the expression of angiogenesis-related factors in the IUGR placenta was significantly reduced, with a concomitant reduction in the expression level of ADORA 2A. Taken together, there is a severe angiogenic failure in the IUGR placenta, accompanied by a decrease in ADORA2A levels.

Example 2 Effect of Water-drinking addition of adenosine 0.1 wt% on placental angiogenesis and IUGR development in female mice

1. Mouse grouping and handling

Selecting 32C 57BL/6J female mice with the age of 7 weeks and 16C 57BL/6J male mice with the age of 8 weeks (purchased from Guangdong medical laboratory animal center, guangdong Fushan, china), feeding maintenance ration (carbohydrate content: 58%, protein content: 18%, fat content: 4.5%) (cooperative medical and biological engineering company) to adapt for one week, and then mating.

On day 21 of breeding, female and male mice are combined in cages according to the proportion of female to male =2:1, the vaginal suppository is checked in the next morning 6. Successful pregnant females were divided into 4 groups according to the principle of consistent body weight: control (Normal), adenosine (Normal + Ado), low protein ration induced intrauterine growth restricted (IUGR) and low protein ration + adenosine (IUGR + Ado). Wherein, the control group and the adenosine group are fed with purified AIN-93G growth and reproduction daily ration (carbohydrate content: 63.95%, protein content: 20.3%, fat content: 7%) during pregnancy, the low-protein daily ration group and the low-protein daily ration plus adenosine group are fed with purified isokinetic low-protein daily ration (carbohydrate content: 77.95%, protein content: 6%, fat content: 7%) during pregnancy, and the daily ration is purchased from Jiangsu cooperative medical bioengineering company. During pregnancy, normal drinking water was given to the control group and the low-protein diet group, and drinking water containing 0.1% adenosine was given to the adenosine group and the low-protein diet + adenosine group, and all the female mice had free drinking water during pregnancy. The test period is 18 days of gestation, pregnant mice are sacrificed at E18.5, and the placenta of the pregnant mice is collected.

2. Sample collection and data recording

As shown in fig. 2A, mice were sacrificed by cervical dislocation 18.5 days of gestation. Uterine-placental-fetal complexes were dissected and weighed. And then, cutting off the uterus from the vaginal end to the ovarian end at one time along the uterine tunica intima side by using a small surgical scissors, pulling out the decidua-coated embryo by using a small forceps, separating the placenta and the fetus, washing in a precooled PBS solution, then sucking off excess water by using filter paper, weighing the weight of the placenta and the weight of a fetal mouse by using balance, and recording the total number of the embryos. And collecting placenta sample, storing the molecular sample at-80 deg.C, and storing the fixed sample in paraformaldehyde solution. Calculating embryo absorption rate of each pregnant mouse, wherein the embryo absorption rate = number of embryos absorbed by the pregnant mouse/total number of embryos.

3. Test results

The test results are shown in fig. 2-4, and the results show that the differences of drinking water and daily ration do not affect the food intake and water intake of the female rat (fig. 2B-C). The low-protein daily ration obviously reduces the body length, the body weight and the placenta efficiency of a fetus, obviously improves the embryo absorption rate and the IUGR incidence rate, and prompts the successful construction of an IUGR model. The supplement of adenosine for drinking water has no significant influence on the development of normal fetal rats, but has significant promotion effect on the development of IUGR fetal rats, including significantly increasing the fetal weight and the fetal body length of the IUGR fetal rats, significantly improving the placental efficiency of the IUGR fetal rats, and significantly reducing the embryo absorption rate and the IUGR incidence rate of the IUGR fetal rats due to the intake of adenosine (fig. 2D-Q).

To explore how adenosine alleviates the occurrence of IUGR. Subsequently, the content change of metabolites in adenosine metabolic pathways in the placenta is detected by using the targeted metabolome, and the content of adenosine and the downstream metabolites inosine and xanthine in the IUGR group is reduced, while the content of adenine is increased, so that the phenomenon can be improved after adenosine is supplemented (fig. 3A-H). In the staining of the placenta sections, the area of the placenta maze area was significantly reduced in the IUGR group, and the area of the blood sinus area was also significantly reduced, and this phenomenon was also significantly reversed after adenosine supplementation (fig. 3I-N).

To further explore the possible underlying mechanisms by which adenosine supplementation improves the development of IUGR, the corresponding molecular markers in the placenta were then examined. The results show that adenosine supplementation significantly ameliorated the decrease in VEGF-A, EGF, ang mRNA levels in the IUGR placenta (fig. 4A), with the same trend shown in the protein levels of Ang, VEGF-A, VCAM (fig. 4B-E). At the same time, ADORA2A mRNA and protein levels were significantly reduced in the IUGR placenta, and adenosine supplementation similarly alleviated this phenomenon (fig. 4F-J). The immunohistochemistry and immunofluorescence results also show that: adenosine supplementation significantly improved the reduction of ADORA2A expression in IUGR placenta, and immunofluorescence simultaneously mapped ADORA2A expression on endothelial cells (fig. 4K-O).

Taken together, the supplementation of water with adenosine may alleviate IUGR by improving the placental angiogenic state, and its mechanism may be linked to the activation of ADORA 2A.

Example 3 mechanism of exogenous adenosine to promote endothelial cell angiogenesis

1. Test materials and methods

Porcine hip endothelial cells (PIECs) were used as an ex vivo model. The angiogenesis capacity of cells is evaluated by adopting tests such as cell scratching, matrigel tube formation and the like, and mechanism exploration is carried out by adopting technologies such as drug intervention, gene expression regulation and the like.

Cell scratch test: when the cells grow to be 75cm²After the bottom of the flask, the cells were digested to prepare a cell suspension, and the cells were counted to obtain PIECs at 5X 10⁵The cells were seeded at a density of one/well in 6-well plates and when 90% of the cells had grown, the plates were scored with a sterile 10 μ L tip and the scored cells were washed away with PBS, control groups were added with serum-free medium, test groups were added with treatment medium and photographed under a microscope and continued at 37 ℃ for 5% CO₂Culturing for 24h in an incubator, and taking pictures by a microscope to record the migration condition of the cells. Cells were randomly picked for 12 fields per well. The migration distance (μm) =0h scratch distance-24 h scratch distance of the cells were analyzed using Image J software.

Matrigel tube test: before the test, matrigel was placed in a 4 ℃ freezer, and 200. Mu.L of the tip and 96-well culture plates were placed in a-20 ℃ freezer. The pre-cooled Matrigel was added to a 96 well plate (placed on ice) with 50. Mu.L per well, avoiding the generation of air bubbles during the procedure, and placed in a cell incubator at 37 ℃ for 30min. Adding 4 multiplied by 10 to each hole after the Matrigel matrix gel is solidified⁴Individual cells, continued at 37 ℃ C. 5% CO₂Incubate for 6h in an incubator and take pictures under a microscope, with 4 fields per well at random. The number of cells in the tube was counted using Image J software.

siRNA transfection: the siRNA was custom-made from Suzhou Ji Ma Biotechnology Inc. and used in conjunction with GP-transfer-Mate transfection reagent according to the instruction, and the final concentration of siRNA in the medium was 100nM. After transfection is finished, western Blot and q-PCR are used for detecting the silencing efficiency of the target gene.

Lentiviral overexpression: lentivirus is customized to Sovia Ji Ma Biotechnology limited, infection conditions are preliminarily searched in a pre-experiment according to the instructions of the company, after suitable culture conditions are screened out, stable transformants are screened out by using a complete culture medium containing 1 mug/ml puromycin, and the stable transformants are frozen and stored for a long time.

2. Test results

The test results are shown in fig. 5 to 10. First, cells were treated with different concentrations of exogenous adenosine. Adenosine concentrations up to 1000 μ M were found to have no significant effect on cell viability (figure 5A). The gap healing and matrigel tube forming tests subsequently showed that adenosine was dose-dependent in promoting the migration and tube forming ability of PIECs and had an adverse effect on PIECs at high concentrations, whereas better effects were achieved at 100. Mu.M concentrations (FIGS. 5B-E). At the same time, the cAMP levels of PIECs were significantly increased upon treatment with 100 μ M exogenous adenosine (fig. 5F).

Then, 100 μ M adenosine concentration was selected for treatment to construct an exogenous adenosine PIECs angiogenesis-promoting model. The results demonstrated the promoting effect of 100 μ M exogenous adenosine on cell migration and angiogenesis (fig. 6A-D), and also showed at the molecular level that adenosine treatment increased the mRNA levels of most angiogenic factors (fig. 6E). At the same time, 100 μ M adenosine treatment was found to significantly promote expression of ADORA2A mRNA only in 4 adenosine receptors (fig. 6F). It was also confirmed that treatment with adenosine also promoted the expression of VEGF-A and adenosine A2 Sub>A receptors (FIG. 6G-I). The above results indicate that exogenous adenosine has the effect of promoting endothelial cell angiogenic ability, and that exogenous adenosine treatment activates the expression of ADORA2A only in 4 adenosine receptors.

To further clarify the effect of ADORA2A on the angiogenic capacity of cells, cells were treated with serum-free medium containing the non-selective adenosine receptor activator NECA (10 μ M) and selective adenosine a2A receptor activator CGS21680 (1 μ M), respectively, for pharmacological intervention on expression of ADORA2A by the cells. The test results show that: both NECA and CGS21680 treatments significantly promoted cell migration (fig. 7a, d) and tube formation ability (fig. 7b, e) without significant effect on cell viability (fig. 7C). The results indicate the ability of ADORA2A activation to indeed promote angiogenesis in endothelial cells.

To further explore the mechanism of action of ADORA2A in regulating cellular angiogenesis, siRNA transfection was used in this experiment to silence ADORA2A expression.

And (3) test results: silencing ADORA2A expression was found to significantly reduce cell migration but had no significant effect on tube formation, whereas siRNA silencing ADORA2A expression significantly reversed the pro-angiogenic phenomenon induced by exogenous adenosine (fig. 8A-D). The RT-PCR results then show: after siRNA transfection, the expression of angiogenesis-related factors is remarkably reduced, and the phenomenon of promoting the expression of the angiogenesis factors caused by exogenous adenosine is also remarkably reversed, wherein the inhibition effect on the expression of Ang mRNA after ADORA2A is silenced is most obvious and reaches more than 90 percent (figure 8E). Western blotting tests also found that the protein levels of ADORA2A, ang, p-Akt and p-Stat3 were significantly reduced after siRNA transfection, and the promotion effect of exogenous adenosine on the expression of these proteins was also reversed (FIGS. 8F-J). Finally, cell line-promoting effects of adenosine were found to be salvaged after 24h treatment with serum-free media containing Akt signaling pathway activator Recilisib (100. Mu.M) and Stat3 signaling pathway activator Colivelin (10. Mu.g/ml), respectively, in the presence of exogenous adenosine and silencing ADORA2A (FIG. 8K-L). The above results show that adenosine promotes endothelial cell angiogenesis by activating ADORA2A and is achieved by activating Akt and Stat3 signaling pathways, possibly associated with altered expression of Ang.

Furthermore, the slow virus infected cell is used for over-expressing the expression of ADORA2A, and the action mechanism of Adenosine/ADORA2A signal contribution pipe is verified. The test results show that: cell migration and tube formation were significantly promoted by lentivirus overexpression of ADORA2A, and further enhanced in the case of exogenous adenosine treatment (fig. 9A-D). Also, overexpression of ADORA2A significantly increased mRNA levels of angiogenesis-related factors in cells, with the Ang mRNA altered most significantly, by about 8-fold (fig. 9E). At the protein level, the protein levels of ADORA2A, ang, p-Akt and p-Stat3 were increased after overexpression of ADORA2A, and the expression level was further increased under treatment with exogenous adenosine (FIGS. 9F-J). The test result further proves that the Adenosine/ADORA2A signal promotes cell angiogenesis by activating downstream Akt and Stat3 signals.

To further elucidate whether the angiopoietic/ADORA 2A capacity is associated with changes in Ang. First, siRNA was used to interfere expression of Ang in cells, and as a result, it was found that silencing Ang expression significantly reduced cell migration and tube formation ability, and at the same time reversed the tube-promoting effect of adenosine (fig. 10A-D). Next, it was found at the protein level that expression of silent Ang did not affect the protein levels of ADORA2A, p-Akt, p-Stat3, whether or not exogenous adenosine treatment was added (FIG. 10E-I). Meanwhile, under the condition of adding adenosine and silencing ADORA2A, the angiogenesis capacity of the cells can be restored after Ang is over-expressed (FIG. 10J-K). Finally, cells were treated with the Akt signaling pathway inhibitors artemisin and Stat3 signaling pathway inhibitor Stattic, respectively, and it was found that both inhibitors reversed the pro-tubular function of adenosine in the presence of adenosine, and that overexpression of Ang could restore the tubular ability of the cells on this basis (fig. 10M-N). Western blotting found that addition of Artemisinin and Static both significantly reduced the protein level of Ang under adenosine treatment (FIG. 10O-P). Test results show that the Adenosine/ADORA2A signal promotes the expression of Ang to exert the capacity of promoting cell angiogenesis by activating Akt and Stat3 signal pathways.

In conclusion, the mechanism of Adenosine's promotion is probably realized by promoting the expression of downstream Stat3, akt signaling pathways and Ang through the Adenosine/ADORA2A signal.

The invention firstly discovers that the IUGR fetal placenta has serious poor angiogenesis state compared with the normal fetal placenta and is accompanied with the down regulation of the expression level of ADORA2A by comparing the angiogenesis and the adenosine expression level of the placenta corresponding to the IUGR fetus in different species. On the basis, the invention further discusses the influence of 0.1% adenosine added in drinking water on placental angiogenesis and IUGR occurrence of pregnant female mice, and further discovers that the supplement of adenosine can improve the placental angiogenesis capability and relieve the IUGR occurrence, and simultaneously discovers that the IUGR placental adenosine metabolism is damaged, the ADORA2A level is reduced, and the supplement of adenosine obviously improves the adenosine metabolic state and re-up-regulates the expression of ADORA 2A. Finally, the invention takes porcine vascular endothelial cells as an in vitro model, researches the mechanism of Adenosine/ADORA2A signal promoting tube, and finds that the mechanism is possibly realized by promoting the expression of downstream Stat3, akt signal channels and Ang. The invention shows that the Adenosine/ADORA2A signal can be used as a powerful means for preventing or relieving IUGR pregnancy outcome, the technology overcomes the defects and shortcomings of the traditional technology, has the advantages of higher safety, better effect and the like, and provides theoretical basis and technical support for the subsequent research and preparation of products for preventing and treating IUGR.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> southern China university of agriculture

Application of <120> ADORA2A gene as molecular marker in preparation of product for preventing or relieving IUGR pregnancy outcome

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agcaacctgc agaacgtcac aaac 24

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tggcaatagc caagaggctg aaga 24

Claims

The application of the ADORA2A gene as a molecular marker in preparing a product for preventing or relieving IUGR pregnancy outcome is characterized in that the adenosine metabolic state in the fetal placenta can be improved by supplementing adenosine, the expression of the ADORA2A gene is up-regulated again, and then the fetal placenta angiogenesis is promoted, so that the occurrence of the IUGR pregnancy outcome is prevented or relieved.
2. The use of claim 1, wherein the product for preventing or ameliorating IUGR pregnancy outcome comprises a product for detecting the expression level of the ADORA2A gene in a fetal placental tissue to be tested, wherein a lower expression level of the ADORA2A gene than in a fetal placental tissue of normal development is indicative of the onset of IUGR in said fetal to be tested.
3. The use of claim 1, wherein the ADORA2A gene prevents or alleviates the occurrence of IUGR pregnancy outcome by promoting fetal placental angiogenesis.
4. The use of claim 1, wherein the IUGR pregnancy outcome comprises: at least one of a decrease in fetal weight and fetal length, a decrease in placental efficiency, an increase in embryo absorption rate, an increase in IUGR rate, a decrease in the area of the maze area of the placenta, and a decrease in the proportion of sinusoid area.
5. The use of claim 1, wherein the product for preventing or alleviating the outcome of IUGR pregnancy comprises: the products of IUGR pregnancy outcome are detected by PCR, RT-PCR, real-time quantitative PCR, DNA sequencing, immunodetection, protein chip, in situ hybridization or gene chip which take genome DNA in the placenta tissue of the fetus to be detected as a template.
6. The use of claim 5, wherein the product for detection of IUGR pregnancy outcome by PCR, RT-PCR, real-time quantitative PCR and DNA sequencing comprises at least one pair of primers for specific amplification of the ADORA2A gene.
7. The use of claim 6, wherein the primers comprise ADORA2A-F and ADORA2A-R and have the nucleotide sequences shown below: ADORA2A-F:5'-AGCAACCTGCAGAACGTCACAAAC-3';

ADORA2A-R：5’-TGGCAATAGCCAAGAGGCTGAAGA-3’。
8. the use of claim 5, wherein the product for the detection of IUGR pregnancy outcome by said immunoassay and protein chip comprises: antibodies, including polyclonal and monoclonal antibodies, that specifically bind to the protein encoded by the ADORA2A gene.
9. The use of claim 5, wherein the product for the detection of IUGR pregnancy outcome by said in situ hybridization or gene chip comprises: a probe that hybridizes to the nucleic acid sequence of the ADORA2A gene.