CN110554175A

CN110554175A - Method for researching function and regulation mechanism of ETR in pregnancy-induced hypertension cell

Info

Publication number: CN110554175A
Application number: CN201910799622.8A
Authority: CN
Inventors: 欧明辉; 徐风森; 纪国欣
Original assignee: Qingdao Municipal Hospital
Current assignee: Qingdao Municipal Hospital
Priority date: 2019-08-28
Filing date: 2019-08-28
Publication date: 2019-12-10

Abstract

the invention discloses a method for researching functions and regulation mechanisms of ETR in pregnancy-induced hypertension cells, and particularly relates to the field of medical research, which comprises the steps of carrying out clinical level research experiments, detecting and analyzing the correlation between plasma ET-1 of pregnancy-induced hypertension patients and inflammatory factors IL-1 beta and IL-18, and carrying out non-transgenic animal level research experiments. According to the invention, clinical experiments, animal experiments, cell experiments, transgenic animal experiments and data of the experiments are developed, the function of ETR in the intimal smooth muscle cell is clearly analyzed and determined by comprehensive experimental results, the expression of ETR in the ET-1 regulation and control vascular endothelial cell under the condition of pregnancy induced hypertension can be clarified, the effect of the expression increase of ETR in the vascular media smooth muscle cell in pregnancy-induced hypertension on the function of blood vessels is clearly determined, and the expression of ETR in the intimal smooth muscle cell is regulated and controlled by the activation of inflammatory corpuscles of the endothelial cell under the condition of pregnancy-induced hypertension is researched.

Description

method for researching function and regulation mechanism of ETR in pregnancy-induced hypertension cell

Technical Field

the invention relates to the field of medical research, in particular to a method for researching functions and regulation mechanisms of ETR in pregnancy-induced hypertension cells.

background

the functional disorder of blood vessels caused by pregnancy-induced hypertension persists after delivery and may lead to increased risk of cardiovascular diseases in these women, and studies in RUPP animal models show that ETR expression in vascular endothelial cells is decreased and ETR expression in smooth muscle cells is increased, wherein ETR in endothelial cells has the function of promoting vasodilation, endothelin has the function of up-regulating intracellular oxidative stress, and the oxidative stress and endothelin can regulate the expression of ETR at the transcriptional level and protein level, respectively, however, the action mechanism in pregnancy-induced hypertension is not clear. Since endothelial cells in the vascular wall and mesangial smooth muscle cells are closely related, it is feasible to study the regulation mechanism of ET-1 on ETR expression in endothelial cells and the mechanism of ETR expression in smooth muscle cells, and there is no research scheme available in the prior art that can study the down-regulation mechanism of ETR expression in endothelial cells in hypertension and cannot clearly determine the up-regulation effect of ETR expression in smooth muscle cells in hypertension.

Therefore, it is necessary to develop a method for studying ETR function and regulation mechanism in the pregnancy induced hypertension cell to solve the above problems.

disclosure of Invention

in order to overcome the above-mentioned drawbacks of the prior art, embodiments of the present invention provide a method for studying the function and regulation mechanism of ETR in pregnancy induced hypertension, which comprises performing clinical experiments, performing animal experiments, performing cell experiments, performing transgenic animal experiments, and collating experimental data, and analyzing the comprehensive experimental results to clarify the function of ETR in mesenteric smooth muscle cells, and can elucidate the expression of ETR in ET-1 regulated vascular endothelial cells under pregnancy induced hypertension, clarify the effect of elevated expression of ETR in mesenteric smooth muscle cells on vascular function, and study the expression of ETR in endothelial cells regulated and controlled by activation of inflammatory corpuscles under pregnancy induced hypertension, so as to solve the problems in the background art.

in order to achieve the purpose, the invention provides the following technical scheme: a method for researching functions and regulation mechanisms of ETR in cells with hypertension syndrome of pregnancy comprises the following specific operation steps:

s1: performing clinical level research experiment to detect and analyze the correlation between plasma ET-1 of the pregnancy-induced hypertension patients and inflammatory factors IL-1 beta and IL-18;

S2: carrying out non-transgenic animal level research experiment, constructing a RUPP animal model, detecting the change of ETR and related passage molecules in intima and media of blood vessels, and constructing the RUPP animal model to analyze the regulation of the ETR on the functions of the blood vessels;

s3: performing a cell level research experiment, researching a regulation mechanism of ET-1 on ETR expression in vascular endothelial cells, detecting the action of ETR on the activity of the vascular endothelial cells, analyzing the action of ET-1 on inflammatory bodies in the vascular endothelial cells, and researching the regulation of inflammatory factors generated by the vascular endothelial cells on the expression of ETR in VSMCs;

s4: carrying out a transgenic animal level research experiment, constructing a transgenic rat model for stably expressing ETR in vascular endothelial cells, constructing a RUPP model on the basis of the rat, detecting blood pressure and plasma ET-1 level by taking a sham operation group as a control, obtaining an aorta and a mesenteric blood vessel after the model is finished, adopting ET-1 stimulation under the condition that an endangium exists, detecting vasoconstriction, adopting acetylcholine stimulation, detecting vasodilation, and obtaining the expression of ETR of an aortic tissue detection intima and a mesenteric;

S5: the experimental data are collated, the results of the experiment are integrated, the function of ETR in mesoderm smooth muscle cells is determined, the regulation and control mechanism of ETR expression in endothelial cells is determined, and whether the ET-1 acts on the endothelial cells to activate the expression of ETR in the upper level smooth muscle cells by verifying a corpuscle signal path is researched.

preferably, the specific operation steps of the clinical level study in S1 are as follows:

S1.1: collecting pregnant woman and normal pregnancy contrast which are clinically diagnosed by a case-contrast research method, screening and grouping pregnant and lying-in woman patients by a hostage standard, and respectively using pregnant hypertension disease lying-in woman and healthy lying-in woman of the same period of childbirth which meet the standard as a pregnant hypertension group and a normal contrast group;

s1.2: collecting samples, taking 3mL of venous blood from pregnant women and pregnant women of the group with hypertension and the group with normal control before parturition, injecting into an Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulant tube, mixing uniformly, centrifuging at 3000r/min for 5-10min at normal temperature to separate blood plasma, taking 0.5mL of supernatant, adding equal volume of 0.4mol/L perchloric acid solution, mixing uniformly, storing in a test tube, centrifuging at 15000r/min4 ℃ for 10min, taking 0.3mL of supernatant, and storing in a refrigerator at-20 ℃ for testing;

S1.3: detecting and recording indexes, adopting an ELISA kit to detect the contents of ET-1, IL-1 beta and IL-18 in the plasma of pregnant women in the pregnancy-hypertension group and the normal control group, and recording detection data;

s1.4: statistical analysis, comparing the difference of ET-1, IL-1 beta and IL-18 content in the plasma of pregnant women in the group with hypertension and the normal control group, and analyzing the correlation between ET-1 and IL-1 beta and IL-18 by using SPSS15.0 statistical software package.

preferably, the specific operation steps of the non-transgenic animal level research in S2 are as follows:

s2.1: RUPP animal model construction, 50 SPF SD rats of 12 weeks old are purchased, raised in a temperature control room with 12h light/dark cycle, and free to eat and drink water, and the RUPP is constructed by reducing the uterine perfusion pressure as follows: on day 14 of pregnancy, rats weighing about 200-;

s2.2, taking a normal pregnant mouse as a control, detecting the blood pressure and the plasma ET-1 level of the model and the control mouse, analyzing whether the model is successfully established or not, taking tissue materials after the model is successfully established, respectively measuring and recording the mean arterial pressure of two groups of rats, collecting urine samples and centrifuging, storing the urine samples in a refrigerator at the temperature of-80 ℃ for later use, simultaneously collecting a plasma sample, sucking CO ₂ into the rats for euthanasia, collecting aortic tissues, fixing one part of the aortic tissues by using 4% formaldehyde, manufacturing paraffin sections, extracting RNA and protein by using one part of the aortic tissues and in vitro researching the blood vessel function by using the other part of the aortic tissues;

s2.3: detecting collected samples, detecting urine protein concentration in urine by a rat albumin ELISA kit, detecting the expression levels of ET-1, IL-1 beta and IL-18 in serum by the rat ELISA kit, detecting the expression of ETR in aorta by a q-PCR method, analyzing whether the expression of ETR is changed at the protein level after transcription or at the transcription level, detecting the expression of ETR, NLRP3, Nrf2, beta-catenin, pro-CASP1, CASP1, pro-IL-1 beta, pro-IL-18 and IL-18 in aorta by a Westernblot method, analyzing the expression of inflammatory corpuscle signal path molecules in aorta and mesenteric blood vessels in a hypertension model caused by pregnancy, dyeing and detecting the thickness of blood vessels, analyzing the function of the pregnancy hypertension on the proliferation of VSMCs (smooth muscle cells) of the blood vessels, staining to detect the apoptosis of the intima of the blood vessel, and analyzing the influence of the gestational hypertension on the apoptosis of endothelial cells of the intima of the blood vessel;

s2.4: performing histological detection, adopting H & E and Masson's staining to detect pathological conditions of aortic tissues, adopting an immunofluorescence experiment to detect the co-localization condition of ETR, Rab-11 and Rab-7 in the aorta if the ETR expression of endothelial cell level is changed only in protein level, analyzing the possibility of the ETR being ubiquitinated and degraded, and detecting the expression of beta-catenin and Nrf2 if the ETR expression of endothelial cell level is changed in RNA level, analyzing the possibility of the transcriptional activity reduction of the ETR;

s2.5: performing vascular function detection, stimulating with ET-1 in the presence of intimal integrity, detecting vasoconstriction, stimulating with acetylcholine, detecting vasodilation, treating with ETAR antagonist BQ-123 and ETR inhibitor BQ-788 respectively in the case of removing intimal, stimulating with ET-1, detecting vasoconstriction, and analyzing the effect of ETR on smooth muscle contraction function.

preferably, the specific operation steps of the cell level research in S3 are as follows:

S3.1: purchasing human aortic endothelial cell HAEC and separating rat primary vascular endothelial cell R-EC respectively, culturing, setting concentration gradient and time gradient ET-1 to treat the endothelial cells, detecting the expression of ETR in the endothelial cells at RNA and protein level, and analyzing whether the regulation of ET-1 on the expression of ETR occurs at the protein level or RNA transcription level;

s3.2: determining the optimal ET-1 treatment concentration and time according to the ETR expression, then stimulating endothelial cells by using ET-1, and detecting the ROS level of the endothelial cells and the expressions of Nrf2 and beta-catenin if the ETR expression changes at the transcription level; then respectively adopting ET-1 stimulation to detect the ROS level and the expression of ETR under the conditions of adopting siRNA to knock down the expression of Nrf2 or beta-catenin, adopting overexpression plasmid to overexpress Nrf2 or beta-catenin and adopting ROS scavenger, analyzing a mechanism that ET-1 regulates the expression of ETR through Nrf2 and analyzing a mechanism that ET-1 regulates the expression of ETR through beta-catenin;

s3.3: co-localization of ETR and Rab-7 was detected if ETR expression was only changed at the protein level; then transiently transfecting a Myc-Ub plasmid carrying a Myc-tagged plasmid of over-expression of a fanin protein in endothelial cells, detecting that ETR is ubiquitinated and modified by an IP experiment under the condition of ET-1 stimulation, and analyzing a mechanism that ET-1 promotes ETR degradation by ubiquitination and modification;

S3.4: respectively adopting siRNA to knock down ETR and overexpression ETR of overexpression plasmid, adopting ET-1 to stimulate vascular endothelial cells, and detecting the activity of the vascular endothelial cells;

s3.5: detecting the expression of NLRP3, pro-CASP1, CASP1, pro-IL-1 beta, pro-IL-18 and IL-18 in endothelial cells by adopting ET-1 stimulation under the conditions of knocking down Nrf2, knocking down beta-catenin, over-expressing Nrf2 by adopting over-expression plasmids and adopting an ROS scavenger respectively, detecting the level of IL-1 beta and IL-18 in supernatant, and analyzing the regulation of ET-1 on the generation of inflammatory corpuscles in the endothelial cells;

s3.6: ET-1 stimulates endothelial cells to prepare conditioned medium, stimulates VSMCs with the conditioned medium, detects nuclear translocation of p65 and expression of ETR in the VSMCs, stimulates the VSMCs with IL-1 beta and IL-18, detects nuclear translocation of p65 and expression of ETR in the cells, then blocks expression of IL-1 beta and IL-18 with antibodies while stimulating the VSMCs with the conditioned medium of endothelial cells, and detects nuclear translocation of p65 and expression of ETR in the cells.

preferably, the specific operation steps of the transgenic animal level study in S4 are as follows:

s4.1: constructing a transgenic rat stably expressing ETR in endothelial cells according to the method of S2.1, adopting doxycycline dox to stimulate and induce ETR conditional expression and ubiquitination site mutation to enable the ETR conditional expression not to be modified by a fantin, and randomly distributing the rat to a RUPP or a sham operation control group;

s4.2: performing rat model verification, obtaining aorta and mesenteric vessels after the model is finished, adopting ET-1 stimulation in the presence of vascular intima to detect vasoconstriction, adopting acetylcholine stimulation to detect vasodilation, simultaneously obtaining aortic tissues of two groups of rats, detecting the expression of intima and media ETR, dyeing to detect the thickness of the media of the blood vessels, and performing blood vessel function detection under the condition that endothelium is finished;

s4.3: analysis of ETR expression in endothelial cells at the level of transgenic rats the regulation of ETR expression in VSMCs of vascular function was investigated.

preferably, in S3.1, the primary vascular endothelial cells and smooth muscle cells are isolated and cultured, and the specific steps include:

s3.1.1: completely separating the aorta of a normal SD rat, removing fat and connective tissues on the surface of the aorta tissue, washing with HBSS for 2 times, and inverting the aorta under a dissecting microscope;

s3.1.2: enzyme digestion culture: adding 3ml of collagenase digestive juice into a 15ml centrifuge tube, digesting for 20min at 37 ℃, and slightly and fully blowing cell digestive juice every 10min during digestion to ensure that cells fall off as much as possible;

s3.1.3: filtering out undigested part of tissue by using a 200-mesh cell sieve, centrifuging for 5min at 1500rpm, removing supernatant, and adding 3ml of complete culture solution into precipitated cells for resuspension;

s3.1.4: digesting the tissue with 0.1% pancreatin for 20min, slightly and fully blowing cell digestive juice every 10min during digestion to ensure that the cells fall off as much as possible, filtering out undigested part of tissue by using a 200-mesh cell sieve, centrifuging at 1500rpm for 5min, removing supernatant, and adding 3ml of complete culture solution into precipitated cells for re-suspension;

s3.1.5, mixing the above two steps of re-suspension culture solution, inoculating into 25cm ² culture flask, culturing at 37 deg.C in 5% CO ₂ incubator for 2h, sucking out the culture medium from the flask, adding 5ml fresh culture medium into the flask, and continuing culturing.

S3.1.6: and (4) EC identification: cells from passage 3 were identified by immunofluorescence staining with CD 31.

The invention has the technical effects and advantages that:

1. By carrying out clinical experiments, animal experiments, cell experiments, transgenic animal experiments and arrangement of experimental data, the function of ETR in the intimal smooth muscle cell is clearly determined by analyzing comprehensive experimental results, the expression of ETR in the ET-1 regulation and control vascular endothelial cells under the condition of hypertension induced by pregnancy can be clarified, the effect of the expression increase of ETR in the vascular media smooth muscle cell in pregnancy-induced hypertension on the function of blood vessels is clearly determined, and the expression of ETR in the intimal smooth muscle cell regulated and controlled by the endothelial cells through the activation of inflammatory corpuscles under the condition of pregnancy-induced hypertension is researched.

drawings

FIG. 1 is a flow chart of the clinical level study experiment of the present invention.

FIG. 2 is a flow chart of the non-transgenic animal level study experiment of the present invention.

FIG. 3 is a flow chart of the cell level study experiment of the present invention.

FIG. 4 is a flow chart of the level study experiment of the transgenic animal of the present invention.

FIG. 5 is a diagram showing ETR function and regulation mechanism simulation according to the experimental results of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

The invention provides a method for researching functions and regulation mechanisms of ETR in a pregnancy-induced hypertension cell, which comprises the following specific operation steps:

further, the specific operation steps of the clinical level study in S1 are as follows:

the nano-ranking standard comprises an entering standard and an excluding standard, wherein the entering standard specifically comprises the following steps:

I conforms to the diagnosis standard of pregnancy-induced hypertension in the guide;

II, the first diagnosis is confirmed, and the previous pregnancy does not have a history of pregnancy-induced hypertension;

III is the pathological obstetric diseases of primary single pregnancy, no pre-placenta, early peeling of placenta, premature rupture of fetal membranes and the like;

IV, primary hypertension, diabetes and other cardiovascular diseases are avoided, and the history of heart, liver and kidney diseases and the history of immune system diseases are excluded;

V can independently complete follow-up data filling and operation informed consent;

The exclusion criteria are specifically:

I, complicating pregnancy complications such as gestational diabetes mellitus, gestational heart disease and the like;

II combined systemic infectious diseases;

III severe liver and kidney insufficiency;

all samples were used and studied under the premise of receiving approval from the ethical committee of the hospital and making informed consent with the subjects;

Further, the specific operation steps of the non-transgenic animal level research in the S2 are as follows:

Further, the specific operation steps of the cell level research in the S3 are as follows:

Further, the concrete operation steps of the transgenic animal level research in S4 are as follows:

Further, in the step S3.1, the primary vascular endothelial cells and smooth muscle cells are isolated and cultured, and the specific operation steps include:

wherein the concentration gradient for stimulating endothelial cells is referenced to: 10^ -10mol/L, 50^ -10mol/L, 10^ -9mol/L, 510^ -9mmol/L, the time gradient reference of the stimulated endothelial cells: and 6h, 12h and 24h, detecting the expression of ETR in endothelial cells at the level of RNA and protein, and determining the optimal ET-1 treatment concentration and time according to the expression of ETR.

collecting blood samples of patients with pregnancy-induced hypertension and normal pregnancy control before delivery by performing clinical experiments, detecting and analyzing the correlation between ET-1 and inflammatory factors IL-1 beta and IL-18 in pregnancy-induced hypertension, performing animal experiments, constructing a RUPP rat model, analyzing the expression change of ETR in aortic vascular cells, the contraction and relaxation of blood vessels, the proliferation of vascular cells by using normal rats as control, analyzing the correlation of the regulation effect of ETR on vascular functions in the RUPP animal model, performing cell experiments, constructing an ETR overexpression plasmid and a siRNA sequence, analyzing the mechanism of regulating ETR expression by ET-1 stimulation, IP-mass spectrometry experiment, and the activation of inflammatory vesicles in endothelial cells, stimulating by IL-1 beta and IL-8, and blocking the effects of IL-1 beta and IL-8 by using small molecules, analyzing the mechanism of inflammatory corpuscles on the expression of ETR in smooth muscle cells, constructing transgenic rats with stable over-expression of ETR, constructing a RUPP rat model, analyzing the expression change of ETR in aortic vascular cells, the contraction and relaxation of blood vessels and the proliferation of vascular cells, researching and analyzing the correlation of the regulation effect of ETR on vascular functions in the RUPP animal model, finally comprehensively completing the experiment, analyzing the experiment data and supplementing the experiment results, clarifying the expression of ETR in ET-1 regulation and control vascular endothelial cells under pregnancy induced hypertension conditions, defining the effect of the expression increase of ETR in the vascular mesomembranous smooth muscle cells in pregnancy induced hypertension on the vascular functions, and researching and controlling the expression of ETR in the intimal smooth muscle cells through the activation of inflammatory corpuscles.

finally, it should be noted that: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.

Claims

1. a method for researching functions and regulation mechanisms of ETR in cells with hypertension of pregnancy, which is characterized in that: the specific operation steps are as follows:

2. the method of claim 1, wherein the function and regulation mechanism of ETR in PIH cells is characterized by: the specific operation steps of the clinical level research in the S1 are as follows:

3. the method of claim 1, wherein the function and regulation mechanism of ETR in PIH cells is characterized by: the specific operation steps of the non-transgenic animal level research in the S2 are as follows:

4. The method of claim 1, wherein the function and regulation mechanism of ETR in PIH cells is characterized by: the specific operation steps of the cell level research in S3 are as follows:

5. The method of claim 1, wherein the function and regulation mechanism of ETR in PIH cells is characterized by: the specific operation steps of the transgenic animal level research in S4 are as follows:

6. the method of claim 4, wherein the function and regulation mechanism of ETR in PIH cells is determined by: in the S3.1, the separation and culture of the primary vascular endothelial cells and the smooth muscle cells comprise the following specific operation steps:

S3.1.5, uniformly mixing the heavy suspension culture solution in the two steps, inoculating the mixture into a 25cm ² culture bottle, culturing for 2 hours in a 5% CO ₂ incubator at 37 ℃, slightly sucking out the culture medium in the bottle, and adding 5ml of fresh culture medium into the bottle to continue culturing;