Construction method and application of chick embryo high-sugar microenvironment model based on chorioallantoic intracavity microinjection

Abstract

The invention belongs to the technical field of basic medicine, and relates to a construction method and application of a chick embryo high-sugar microenvironment model based on microinjection in a chorioallantoic cavity, glucose is injected into the chorioallantoic cavity on the 5th day and the 7th day of egg incubation to construct the chick embryo high-sugar microenvironment model, the maternal effect can be eliminated, and the chick embryo high-sugar microenvironment model is obtained. The method can be used for independently researching the influence of the high-glucose microenvironment on the embryonic development, is low in cost, easy to operate, easy to achieve a relatively high experimental sample size, relatively large in embryos and convenient to directly observe and operate, and can become a powerful tool for researching the influence of the high-glucose microenvironment on the embryonic development and filial generation health.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of basic medicine, and relates to a method for establishing a chicken embryo high-sugar microenvironment model based on chorioallantoic cavity injection technology, and a method for carrying out related basic medical research based on the model. BACKGROUND

[0002] Gestational diabetes mellitus (definition) is one of the most common complications during pregnancy. Gestational diabetes mellitus not only poses a threat to maternal health, but also has a profound impact on the developing fetus. One of the main direct impacts is that the fetus is exposed to a high-sugar microenvironment, which not only increases the risk of direct complications in the offspring of gestational diabetes mellitus, such as macrosomia, premature birth, birth trauma, shoulder dystocia, neonatal hypoglycemia, etc., but more importantly, according to the classic Developmental Origins of Health and Disease (DOHaD) theory, the microenvironment during embryonic development can affect the fetus during pregnancy, after birth, and even long-term health in adulthood. Existing epidemiological studies and animal experiments have found that the offspring of gestational diabetes mellitus face a variety of problems after birth, such as significantly increased risks of obesity, diabetes, and cardiovascular diseases, metabolic system problems, respiratory system problems, etc. The current common experimental model for gestational diabetes mellitus animal research is to use Streptozocin (STZ) to treat pregnant mice to induce diabetes in pregnant mice, thereby achieving an effect similar to gestational diabetes mellitus. However, STZ is a classic drug that destroys pancreatic beta cells to induce diabetes, which is significantly different from the real occurrence of gestational diabetes mellitus. It is generally recognized that this method causes the effect of general diabetes, and gestational diabetes mellitus and general diabetes differ greatly in pathogenesis, symptoms, treatment, and prognosis. STZ may also directly affect the embryo during modeling, interfering with the accurate observation of the effects on the offspring.

[0003] Currently, there is no report on directly constructing an embryo model in a high-sugar environment. The present application fills this gap and provides a powerful tool for gestational diabetes mellitus-related research. SUMMARY

[0004] The purpose of the present application is to overcome the problem that the existing gestational diabetes mellitus model relies on Streptozocin for modeling in pregnant mice and cannot directly simulate the high-sugar microenvironment of the embryo, and to provide a method for establishing a chicken embryo high-sugar microenvironment based on micro-injection into the chorioallantoic cavity, which excludes maternal effects and provides a powerful tool for studying the effects of high-sugar microenvironments on embryonic development and offspring health.

[0005] To achieve the above objectives, the technical solution adopted by this invention is to provide a method for constructing a high-glucose microenvironment model of chicken embryos based on intrachorioallantoic microinjection. This method involves injecting glucose into the chorioallantoic cavity on the 5th and 7th days of egg incubation to construct the high-glucose microenvironment model of chicken embryos, wherein the glucose injection dosage is 480 mg / kg. The specific steps are as follows:

[0006] (1) Take out the fertilized eggs that have been incubated for 5 days from the incubator, quickly candle the eggs in a dark room, mark the location of the air cell on the eggshell with a pencil, and make a hole in the location of the air cell on the eggshell with ophthalmic tweezers to form an opening with a diameter of 2-3cm.

[0007] (2) Mark the injection point: The glucose injection point is located 1-1.5 cm from the center of the ventricular membrane.

[0008] (3) Injecting glucose: After sterilizing the microsyringe, draw up the sterile glucose solution. At the glucose injection point, gently insert the microsyringe into the chorionic allantoic cavity at a 30-degree angle to the membrane. After injection, keep it still and allow the glucose solution to diffuse fully before withdrawing the microsyringe in a straight line. Do not stir. Seal the eggshell opening with transparent tape.

[0009] (4) When the fertilized eggs continue to be incubated until the 7th day, glucose is injected again according to the method in step (3), and the high sugar microenvironment chicken embryo model is established.

[0010] The injection volume of the sterile glucose solution described in this invention is 1.5 μL / 10g egg weight.

[0011] The concentration of the sterile glucose solution described in this invention is 3.2 g / mL.

[0012] The sterile glucose solution described in this invention is obtained by dissolving glucose powder in physiological saline.

[0013] The method for marking injection points according to the present invention is as follows: use a water-based marker to gently make a cross mark on the inner membrane of the air chamber with the center of the inner membrane of the air chamber as the center point, and the endpoint of the cross mark extends to the edge of the opening of the eggshell; any endpoint of the cross mark can be used as a glucose injection point.

[0014] When injecting glucose as described in this invention, the microsyringe enters the chorionic allantoic cavity to a depth of 2-3 mm.

[0015] This invention can verify whether a high-glucose microenvironment has been successfully constructed by detecting the glucose level of the chorionic allantoic fluid in the chorionic allantoic cavity before and after two glucose injections. Specifically, the chorionic allantoic fluid is extracted and its glucose level is detected before the first injection, 1 hour, 2 hours and 1 day after the first injection, and 1 hour and 2 hours after the second injection. The two injections significantly increased the glucose level in the chorionic allantoic cavity, indicating that the high-glucose microenvironment was successfully established.

[0016] The specific method for extracting chorionic allantoic fluid according to the present invention is as follows:

[0017] (1) Mark the extraction point: The extraction point is 1-1.5cm away from the center of the inner membrane of the air chamber. The angle between the line (straight line) connecting the extraction point and the center of the inner membrane of the air chamber and the line (straight line) connecting the injection point and the center of the inner membrane of the air chamber is 90 degrees.

[0018] (2) After sterilizing the microsyringe, at the extraction point, the microsyringe is inserted perpendicularly to the membrane into the chorionic allantoic cavity to a depth of 2-3 mm. 2 microliters of chorionic allantoic fluid are gently extracted and withdrawn in a straight line. The baseline glucose level of the chorionic allantoic fluid is detected using a blood glucose meter or a glucose quantitative kit.

[0019] The chorioallantoic cavity (also known as the allantoic cavity) described in this invention contains chorioallantoic fluid (also known as allantoic fluid).

[0020] This invention also provides the application of the chicken embryo high-sugar microenvironment model constructed by the above method, for studying the effects of the high-sugar microenvironment on embryonic development and offspring health.

[0021] The method described in this invention does not require streptozotocin for modeling. It directly uses glucose to generate a high-glucose microenvironment in the developing embryo. Since the chicken embryo grows independently of the mother, the maternal effect can be eliminated, and the impact of the high-glucose microenvironment on embryonic development can be studied independently. Subsequently, the experiment can be stopped at the embryonic stage to observe the effect of the high-glucose microenvironment on embryonic development, or the animals can be hatched to observe the impact of the high-glucose microenvironment on offspring health during the embryonic stage. It is a good supplement to existing gestational diabetes models.

[0022] The beneficial effects of this invention are: it successfully establishes a method for creating a high-glucose microenvironment in chicken embryos based on intracavitary microinjection of chorionic allantoic fluid, which can eliminate maternal effects and study the effects of the high-glucose microenvironment on embryonic development independently; the construction method of this invention is low-cost, easy to operate, and easy to achieve a high experimental sample size, and the embryos are relatively large, making it easy to observe and operate directly, and can become a powerful tool for studying the effects of the high-glucose microenvironment on embryonic development and offspring health. Attached Figure Description

[0023] Figure 1 This is a top view schematic diagram of the intracavitary microinjection mode of chicken embryo chorionic allantoic cavity involved in the present invention.

[0024] Figure 2 The diagram shows an actual experimental example of intracavitary microinjection of chicken embryo chorionic allantoic fluid according to the present invention. In A, glucose solution is injected by inserting the needle at a 30-degree angle; in B, chorionic allantoic fluid is extracted at a 90-degree angle at a predetermined extraction point to detect glucose levels by inserting the needle.

[0025] Figure 3This invention relates to a time-varying curve of glucose levels in chorioallantoic fluid, where Control represents the control group and High glucose represents the high glucose microenvironment group. Data are expressed as mean ± standard deviation. N=4 / group.

[0026] Figure 4 This document presents the experimental results of Example 2 of the present invention regarding the health effects of a high-sugar microenvironment on embryonic development. A shows a Masson staining image, with a scale bar representing 50 micrometers; B is a statistical graph. N=3 / group, and data are expressed as mean ± standard deviation. *: Significant difference compared to the control group, P<0.05. #: Significant difference compared to the exhaust gas group, P<0.05. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0028] Example 1:

[0029] This embodiment relates to a method for constructing a high-glucose microenvironment model of chicken embryos based on intracavitary microinjection of chorionic allantoic fluid. The specific steps are as follows:

[0030] (1) Experimental preparation: Take normal fertilized eggs and incubate them under standard incubation conditions until day 5;

[0031] (2) Prepare a sterile high-concentration glucose solution. The specific steps are as follows:

[0032] (a) Weigh 1.60 g of glucose powder, add 500 μL of physiological saline, and incubate in a water bath at 60 °C until completely dissolved to obtain a glucose solution of 3.2 g / mL.

[0033] (b) Draw the completely dissolved glucose solution into a disposable syringe, then insert an aqueous filter membrane and slowly filter the solution into a 1.5 mL centrifuge tube;

[0034] (3) Take the egg 1 out of the incubator and quickly candle it in the dark. Mark the location of the air cell on the eggshell with a pencil (Note: the location of the air cell will change as incubation progresses, so it should not be marked in advance). Use ophthalmic tweezers to make a hole in the center of the air cell on the eggshell and expand it outwards, making sure not to damage the inner membrane of the air cell, to form an opening 2 with a diameter of about 2-3 cm.

[0035] (4) Use a water-based marker to gently mark a cross on the air cell membrane at the opening 2 of the eggshell, with the center of the air cell membrane as the center point. The endpoints of the cross mark extend to the edge of the opening 2. Any three endpoints of the cross mark are respectively used as two chorionic allantoic fluid extraction points 4 and one glucose injection point 5. The line connecting the two chorionic allantoic fluid extraction points is on the cross mark line. The two chorionic allantoic fluid extraction points are perpendicular to the glucose injection point on the cross mark. That is, the endpoints on both sides of the glucose injection point are chorionic allantoic fluid extraction points.

[0036] (5) After sterilizing the microsyringe by flaming with an alcohol lamp, take one end of the cross mark (about 1.5 cm from the center of the membrane inside the air chamber) as the chorionic allantoic fluid extraction point. At this extraction point, gently insert the microsyringe perpendicularly into the chorionic allantoic cavity to a depth of about 2 mm (the depth can be pre-marked on the syringe needle). Gently extract 2 μL of chorionic allantoic fluid and withdraw it in a straight line. Use a blood glucose meter or glucose quantification kit to detect the baseline glucose level of the chorionic allantoic fluid. The results are as follows: Figure 3 As shown;

[0037] (6) After sterilizing the microsyringe by burning with an alcohol lamp, draw up the glucose solution (1.5ul / 10g egg weight). On the cross mark, take the endpoint that is 90 degrees from the point where the chorionic allantoic fluid was drawn in step (5) as the glucose injection point. At the glucose injection point, insert the microsyringe into the chorionic allantoic cavity at a 30-degree angle to the membrane, facing outwards, to a depth of about 3mm (the depth can be marked on the needle of the syringe beforehand). Inject gently, and after injection, hold for about 10 seconds to allow the glucose solution to fully diffuse before withdrawing in a straight line without stirring. Seal the opening of the eggshell with transparent tape.

[0038] (7) Glucose levels were measured 1 hour, 2 hours and 1 day after glucose injection. The transparent tape was removed, and the microsyringe was sterilized by flaming with an alcohol lamp at the chorioallantoic fluid extraction point (the endpoint at a 90-degree angle to the glucose injection point (approximately 1.5 cm from the center of the endocardial membrane)). The microsyringe was then gently inserted perpendicularly into the chorioallantoic cavity to a depth of approximately 2 mm (the depth can be pre-marked on the needle). Two microliters of chorioallantoic fluid were gently extracted, withdrawn in a straight line, and sealed again with transparent tape. The glucose level of the chorioallantoic fluid was measured using a blood glucose meter or a glucose quantification kit. The results are as follows: Figure 3 As shown; the two endpoints at a 90-degree angle to the injection point should be used alternately to avoid repeated and frequent damage to the same spot, which could increase the mortality rate.

[0039] (8) When the fertilized eggs are incubated for 7 days, glucose is injected again at the same injection point as in step (6). Then, chorionic allantoic fluid is drawn 1 hour and 2 hours after the injection to measure the glucose level. The results are as follows: Figure 3 As shown, the high-sugar microenvironment chicken embryo model has been successfully established. It can be cultured further, or the embryos can be harvested immediately for various assays.

[0040] Figure 3 The glucose levels of chorionic fluid extracted on day 5 of fertilized egg incubation (step 5), 1 hour and 2 hours after injection on day 5, and 1 hour and 2 hours after injection on days 6 and 7 are shown. The glucose level changes over time are plotted. It can be seen that the two injections significantly increased the glucose level in the chorionic membrane, indicating that the high-glucose microenvironment was successfully established.

[0041] Note: Glucose level measurement is to verify the effectiveness of the high-glucose microenvironment. However, collecting chorionic villus fluid inevitably increases embryo mortality. After conducting 1-2 batches of experiments to confirm the establishment of the high-glucose microenvironment, it is no longer necessary to collect chorionic villus fluid for verification every time. Instead, glucose injections can be performed only on days 5 and 7 of egg incubation. When injecting glucose only, all four endpoints of the cross-shaped marker can be used as injection sites.

[0042] This invention utilizes the natural and easily manipulated chorionic membrane structure of chicken embryos and employs embryo microinjection technology to create a high-glucose microenvironment in the embryo caused by glucose alone. This allows for the establishment of a model of the impact of the high-glucose microenvironment on embryonic development, which can be used to study various health effects in offspring of gestational diabetes.

[0043] Example 2:

[0044] This embodiment relates to a study on the health of chicken embryo development in the high-sugar microenvironment established in Example 1. The experiment was divided into four groups: a normal control group (incubated with normal chicken embryos), a high-sugar group (incubated with chicken embryos in the high-sugar microenvironment constructed according to the method in Example 1), an exhaust gas group (normal chicken embryos were incubated after being exposed to exhaust gas through the air chamber), and a high-sugar + exhaust gas group (chicken embryos in the high-sugar microenvironment were incubated after being exposed to exhaust gas through the air chamber). The specific method of air chamber inhalation exposure was as follows: At 18-19 days of chicken embryo incubation, an air chamber inhalation exposure experiment was conducted using the method described in the authorized invention patent "A Method for Exposing Chicken Embryos to Diesel Engine Exhaust Gas" (patent number ZL 201910778597.5), inducing the incubating chicken embryos to inhale diluted (50% concentration) diesel engine exhaust gas. The chicks from all four groups were raised normally for one month after hatching, then sacrificed, and lung tissue was collected for Masson staining. Quantitative analysis was performed using ImageJ with blue staining, and statistical analysis was conducted using SPSS 17.0. Figure 4 As shown.

[0045] from Figure 4 It can be seen that exposure to a high-sugar microenvironment itself does not cause pulmonary fibrosis. However, animals exposed to a high-sugar microenvironment during the embryonic period and then inhaling diesel exhaust gas at birth can develop more severe pulmonary fibrosis than animals not exposed to a high-sugar microenvironment. This indicates that the high-sugar microenvironment in chicken embryos significantly enhances the sensitivity of hatched animals to the pulmonary toxicity of diesel exhaust gas. The high-sugar microenvironment model has high research value.

[0046] In summary, the intracellular glucose microinjection technique of the chorioallantoic membrane described in this invention can achieve a high-glucose microenvironment during embryonic development at low cost and high efficiency, thereby simulating the environment of the embryo in gestational diabetes mellitus. This technique can be used to study the health risk of offspring with gestational diabetes mellitus and is an innovative and promising new research model.

Claims

1. A method for constructing a chicken embryo high glucose microenvironment model based on villous allantoic cavity microinjection, characterized in that, The high glucose microenvironment model of chick embryo is constructed by injecting glucose into the chorioallantoic cavity at the 5th day and the 7th day of the egg incubation.

2. The method for constructing chicken embryo high sugar microenvironment model based on villi allantoic cavity microinjection according to claim 1, characterized in that, The specific steps are as follows: (1) Take the fertilized eggs incubated for 5 days from the incubator, quickly take the eggs to the dark room, mark the air chamber position on the egg shell with a pencil, and make a hole on the air chamber position of the egg shell with an ophthalmic forceps to form an opening with a diameter of 2-3 cm; (2) Mark the injection point: mark the glucose injection point at 1-1.5 cm from the center of the air chamber membrane; (3) Inject glucose: after sterilizing the micro-syringe, draw the sterile glucose solution, and gently insert the micro-syringe into the chorioallantoic cavity at the glucose injection point at an angle of 30 degrees with the membrane, then inject, keep still after injection, and then withdraw the micro-syringe in a straight line after the glucose solution is fully diffused, and do not stir; seal the opening of the egg shell with transparent tape; (4) When the above fertilized eggs are incubated to the 7th day, glucose is injected again according to the method of step (3), and the high glucose microenvironment chick embryo model is completed.

3. The method for constructing chicken embryo high sugar microenvironment model based on villi allantoic cavity microinjection according to claim 2, characterized in that, The injection amount of the sterile glucose solution is 1.5 ul / 10 g of egg weight; the concentration of the sterile glucose solution is 3.2 g / mL.

4. The method for constructing chicken embryo high sugar microenvironment model based on villi allantoic cavity microinjection according to claim 2, characterized in that, The sterile glucose solution is obtained by dissolving glucose powder in normal saline.

5. The method for constructing chicken embryo high sugar microenvironment model based on villi allantoic cavity microinjection according to claim 2, characterized in that, The method for marking the injection point is as follows: make a cross mark on the air chamber membrane with the center of the air chamber membrane as the center, and extend the endpoints of the cross mark to the edge of the opening of the egg shell; Any endpoint of the cross mark can be used as the glucose injection point.

6. The method for constructing chicken embryo high sugar microenvironment model based on villi allantoic cavity microinjection according to claim 2, characterized in that, When injecting glucose, the depth of the micro-syringe into the chorioallantoic cavity is 2-3 mm.

7. The method for constructing chicken embryo high sugar microenvironment model based on villi allantoic cavity microinjection according to claim 2, characterized in that, The success of the construction of the high glucose microenvironment can be verified by detecting the glucose level in the chorioallantoic fluid before and after the two injections, specifically, the glucose level in the chorioallantoic fluid is detected before the first injection, 1 hour, 2 hours and 1 day after the first injection, and 1 hour, 2 hours after the second injection, and the glucose level in the chorioallantoic cavity is significantly increased after the two injections, indicating that the high glucose microenvironment is successfully established.

8. The method for constructing chicken embryo high sugar microenvironment model based on villi allantoic cavity microinjection according to claim 7, characterized in that, The specific method for extracting the chorioallantoic fluid is as follows: (1) Mark the extraction point: the extraction point is 1-1.5 cm from the center of the air chamber membrane, and the angle between the line connecting the extraction point and the center of the air chamber membrane and the line connecting the injection point and the center of the air chamber membrane is 90 degrees; (2) After sterilizing the micro-syringe, gently insert the micro-syringe into the chorioallantoic cavity at the extraction point at a right angle with the membrane to a depth of 2-3 mm, gently extract 2 microliters of chorioallantoic fluid, and withdraw in a straight line, and detect the glucose baseline level of the chorioallantoic fluid with a blood glucose meter or a glucose quantitative kit.

9. Use of the chicken embryo hyperglycemic microenvironment model constructed according to the construction method of any one of claims 1-8, characterized in that, The high glucose microenvironment is used to study the effects of high glucose microenvironment on embryonic development and offspring health.

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