Establishment method of progesterone non-gene anti-oxidative stress model

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for establishing a progesterone non-gene anti-oxidative stress model. The method comprises the following steps: centrifuging in-vitro blood; centrifuging and then collecting lower-layer red blood cell precipitate to obtain red blood cells; the method comprises the following steps: taking red blood cells, preparing a red blood cell suspension with a volume percentage of 5% by using PBS, adding progesterone and 30mmol / L-80mmol / L of azobis (isobutylamidine hydrochloride) into the red blood cell suspension, and finally obtaining a red blood cell suspension containing the progesterone and the azobis (isobutylamidine hydrochloride); heating and centrifuging; centrifuging, taking a supernatant, measuring the absorbance of 535-545 nm, and recording the absorbance as A1 '; taking red blood cells, and preparing red blood cell suspension which only contains 5 percent by volume of the red blood cells by using PBS (Phosphate Buffer Solution); performing centrifugation; the method comprises the following steps: taking a progesterone sample as a raw material, centrifuging, taking a supernatant, measuring absorbance of 535nm-545nm, recording the absorbance as A2 ', and calculating a red blood cell hemolysis rate according to a percentage value of A1' and A2 'to obtain the progesterone non-gene anti-oxidative stress mode.The method has the advantages that the red blood cells are used for the first time, and the progesterone non-gene anti-oxidative stress model is established.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for establishing a progesterone non-genetic anti-oxidative stress model. Background Art

[0002] Progesterone is a sex hormone mainly secreted by the ovaries, which plays an important role in the development of the endometrium and the maintenance of pregnancy. Existing studies have found that progesterone can not only be secreted by the ovaries, but also synthesized locally by nerve cells, glial cells, etc. in the nervous system. Progesterone plays an important role as a neuromodulator. At the same time, in oxidative stress damage, progesterone also plays an antioxidant role, thereby playing a neuroprotective role. The anti-oxidative stress effect of progesterone can be exerted through two pathways. The first pathway is through the slow action of cytoplasmic receptors to regulate the expression of related genes; the second pathway is through the rapid action of non-genes. In order to study the anti-oxidative stress effect of progesterone, we can study it by establishing an anti-oxidative stress model.

[0003] The existing research methods of progesterone antioxidant stress model include in vitro oxidative stress model and in vivo oxidative stress model. Among them, the main detection index of the in vitro oxidative stress model is the content of various reactive oxygen species in the reaction system. The in vivo oxidative stress model mainly detects the content of various reactive oxygen species or the activity of the antioxidant system in the body after a certain antioxidant product is given to the body. However, the progesterone antioxidant stress model established by the above two methods for establishing the progesterone antioxidant stress model has the problem that it cannot objectively and truly reflect the non-genetic antioxidant stress ability of progesterone. Summary of the invention

[0004] In order to solve the problem that the progesterone antioxidant stress model established by the method for establishing the progesterone antioxidant stress model in the prior art cannot objectively and truly reflect the non-genetic antioxidant stress ability of progesterone, in order to achieve the above purpose, the present invention adopts the following technical scheme.

[0005] The present invention provides a method for establishing a non-genetic anti-oxidative stress model of progesterone. The present invention first constructs an in vitro oxidative stress reaction system, and then adds red blood cells with strong antioxidant capacity of the body to the system, so as to reflect the oxidative stress with the degree of damage to the red blood cells; at the same time, the antioxidant effect of progesterone as an effective antioxidant drug is verified, and it is determined that this system can well provide an in vitro anti-oxidative stress model. The progesterone anti-oxidative stress model established by the method for establishing a non-genetic anti-oxidative stress model of progesterone provided by the present invention can reflect the non-genetic anti-oxidative stress capacity of progesterone in an observable and real way, which solves the problem that the progesterone anti-oxidative stress model established by the method for establishing a progesterone anti-oxidative stress model in the prior art cannot objectively and truly reflect the non-genetic anti-oxidative stress capacity of progesterone.

[0006] The method for establishing the progesterone non-genetic antioxidant stress model specifically comprises the following steps:

[0007] The ex vivo blood is centrifuged; after centrifugation, the red blood cell sediment in the lower layer is collected to obtain red blood cells.

[0008] Take red blood cells, prepare a 5% volume percentage red blood cell suspension with PBS, add 1 nmol / L to 100 nmol / L progesterone and 30 mmol / L to 80 mmol / L azobisisobutylamidine hydrochloride (AAPH) to the red blood cell suspension, and finally obtain a red blood cell suspension containing progesterone and azobisisobutylamidine hydrochloride; heat the red blood cell suspension containing progesterone and azobisisobutylamidine hydrochloride, and centrifuge; take the supernatant after centrifugation, and measure the absorbance at 535 nm to 545 nm, which is recorded as A1'.

[0009] Take red blood cells and prepare a 5% volume percentage red blood cell suspension containing only red blood cells with PBS; heat the 5% volume percentage red blood cell suspension containing only red blood cells for 30 minutes to 180 minutes, and then centrifuge; take the supernatant after centrifugation, and measure the absorbance at 535nm to 545nm, which is recorded as A2'.

[0010] According to the percentage of A1' and A2', the hemolysis rate of red blood cells was calculated to obtain the non-genetic antioxidant stress model of progesterone.

[0011] Preferably, the isolated blood is isolated carotid artery blood. Among them, isolated carotid artery blood is rich in mature red blood cells. Mature red blood cells have functions such as transportation, regulation, and defense, and are also natural anucleated cells in the body. At the same time, red blood cells come into contact with blood in the body circulation and produce a variety of free radicals. Since red blood cells are rich in various antioxidant enzymes, they can effectively protect red blood cells from damage by free radicals, thereby ensuring the structure and function of red blood cells.

[0012] Due to these characteristics of erythrocytes and the ease of obtaining and preparing erythrocytes, erythrocytes become the best model for studying the anti-oxidative stress effect of progesterone in the present invention.

[0013] Preferably, the conditions for the in vitro blood centrifugation are: 2500 rpm to 3500 rpm, and centrifugation for 4 min to 6 min.

[0014] Preferably, the concentration of PBS is 0.005-0.015 M, and the pH is 7.3-7.5.

[0015] Preferably, the concentration of the progesterone is 1.5 nmol / L to 99 nmol / L.

[0016] Preferably, the concentration of the progesterone is any one of 1 nmol / L, 10 nmol / L, 30 nmol / L, 50 nmol / L and 100 nmol / L.

[0017] Preferably, the heating time is 30 min to 180 min.

[0018] Preferably, A1' and A2' measure the absorbance at 540 nm.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a method for establishing a progesterone anti-oxidative stress model. In the method for establishing a progesterone anti-oxidative stress model provided by the present invention, the ex vivo blood is first centrifuged to obtain red blood cells, and then the red blood cells are configured into a 5% volume percentage red blood cell suspension containing different concentrations of progesterone and 50mmol of azobisisobutylamidine hydrochloride through PBS, and then the absorbance is measured to calculate the red blood cell hemolysis rate. The present invention uses red blood cells for the first time to establish a non-genetic anti-oxidative stress model of progesterone. The progesterone anti-oxidative stress model established by the method for establishing a non-genetic anti-oxidative stress model of progesterone provided by the present invention can reflect the non-genetic anti-oxidative stress ability of progesterone in an observable and real way, solving the problem that the progesterone anti-oxidative stress model established by the method for establishing a progesterone anti-oxidative stress model in the prior art cannot objectively and truly reflect the non-genetic anti-oxidative stress ability of progesterone. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure shows the effect of pre-incubation with different concentrations of azobisisobutylamidine hydrochloride on hemolysis of erythrocytes in the present invention.

[0022] Figure 2 The figure shows the effect of different concentrations of progesterone on the hemolysis of red blood cells caused by 50 mmol of azobisisobutylamidine hydrochloride in the present invention. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0024] The rats used in the following examples were 8-week-old, SPF male SD rats, 200±20 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2012-0001.

[0025] Example 1

[0026] A method for establishing a progesterone non-genetic antioxidant stress model comprises the following steps:

[0027] S1. After the rats were anesthetized with 10% wt chloral hydrate at a ratio of 1 mL / 100 g of the volume of chloral hydrate to the mass of the rats, 5 mL of blood was collected through carotid artery cannula using a heparin anticoagulation blood collection tube to obtain ex vivo blood.

[0028] S2. Centrifuge the ex vivo blood obtained in S1 at 3000 rpm for 5 min, discard the supernatant, collect the lower red blood cell pellet, suspend the lower red blood cell pellet in 10 mL of 0.01 M PBS, centrifuge again, discard the supernatant, collect the lower red blood cell pellet, suspend the red blood cells in the obtained lower pellet with PBS again, wash it twice, and obtain a red blood cell solution.

[0029] Among them, the pH of PBS is 7.4.

[0030] S3. Take the red blood cell solution obtained in S2, prepare it into a 5% volume percentage red blood cell suspension with PBS, add different concentrations of azobisisobutylamidine hydrochloride to the red blood cell suspension, and finally obtain a red blood cell suspension containing azobisisobutylamidine hydrochloride; put the red blood cell suspension containing azobisisobutylamidine hydrochloride in a 37°C water bath, take 200 μL at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively, then centrifuge, take the supernatant, and measure the absorbance of the solution at 540 nm at the above-mentioned different time points, respectively, and record it as A1.

[0031] The different concentrations of azobisisobutylamidine hydrochloride are 10mmol / L, 30mmol / L, 50mmol / L, 80mmol / L and 100mmol / L respectively. The English abbreviation of azobisisobutylamidine hydrochloride is AAPH.

[0032] At the same time, PBS was used to prepare a 5% volume percentage red blood cell suspension containing only red blood cells, and it was placed in a 37°C water bath. Similarly, 200 μL was taken at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively, and 1.8 mL of pure water was added respectively. The suspension was centrifuged and the supernatant was taken. The absorbance at 540 nm of the solution at the above-mentioned different time points was measured respectively, and recorded as A2.

[0033] S4. Calculate the first hemolysis rate according to the first hemolysis rate=A1 / A2*100.

[0034] S5. Take the red blood cells obtained in S2, prepare a 5% volume percentage red blood cell suspension with PBS, add different concentrations of progesterone and 50 mmol of azobisisobutylamidine hydrochloride to the red blood cell suspension, and finally obtain a red blood cell suspension containing progesterone and azobisisobutylamidine hydrochloride; place the red blood cell suspension containing progesterone and azobisisobutylamidine hydrochloride in a 37°C water bath, take 200 μL at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively, then centrifuge, take the supernatant, and measure the absorbance of the solution at 540 nm at the above-mentioned different time points, respectively, and record it as A1'.

[0035] Among them, the different concentrations of progesterone are 0, 1nmol / L, 10nmol / L, 30nmol / L, 50nmol / L, and 100nmol / L.

[0036] At the same time, a 5% volume percentage red blood cell suspension containing only red blood cells was prepared and placed in a 37°C water bath. Similarly, 200 μL was taken at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, 1.8 mL of pure water was added, and the suspension was centrifuged. The supernatant was taken and the absorbance at 540 nm of the solution at the above-mentioned different time points was measured, recorded as A2'.

[0037] S6. Calculate the second hemolysis rate according to the second hemolysis rate=A1' / A2'*100.

[0038] It should be noted that the first hemolysis rate and the second hemolysis rate are both hemolysis rates of red blood cells, and both can reflect the degree of hemolysis of red blood cells. Among them, the first hemolysis rate is the hemolysis rate of red blood cells caused by different concentrations of azobisisobutylamidine hydrochloride; the second hemolysis rate is the hemolysis rate caused by different concentrations of progesterone and 50mmol / l azobisisobutylamidine hydrochloride.

[0039] Experimental results:

[0040] 1. After incubation at 37℃ for 1.5h, the hemolysis rate of red blood cells caused by different concentrations of azobisisobutylamidine hydrochloride

[0041] After incubation at 37℃ for 1.5h, compared with the control group (0mmol / L azobisisobutyramidine hydrochloride concentration), the difference between different concentrations of azobisisobutyramidine hydrochloride (10mmol / L, 30mmol / L, 50mmol / L, 80mmol / L, 100mmol / L) could cause different degrees of damage to erythrocytes, leading to different degrees of hemolysis of erythrocytes, and the differences were statistically significant (P<0.05): the hemolysis rate of the 10mmol / L azobisisobutyramidine hydrochloride group was (6.51±2.27)%; the hemolysis rate of the 30mmol / L azobisisobutyramidine hydrochloride group was (26.43±5.36)%; the hemolysis rate of the 50mmol / L azobisisobutyramidine hydrochloride group was (14.87±13.70)%; the hemolysis rate of the 10mmol / L azobisisobutyramidine hydrochloride group was (21.30±15.71)%; the hemolysis rate of the 50mmol / L azobisisobutyramidine hydrochloride group was (3.20±1.53)%. The hemolysis rate of group H was (65.11±6.51)%; the hemolysis rate of 80mmol / L azobisisobutyramidine hydrochloride was (97.15±2.91)%; the hemolysis rate of 100mmol / L azobisisobutyramidine hydrochloride was (99.25.11±1.29)%.

[0042] 2. Hemolysis of red blood cells caused by 50mmol / lAPPH after incubation for different time periods

[0043] When the concentration of azobisisobutylamidine hydrochloride is 50mmol / L, the effects of different incubation times at 37℃ on the hemolysis rate of red blood cells are as follows:

[0044] When the incubation time was 0 min, the hemolysis rate was (0±0)%; when the incubation time was 30 min, the hemolysis rate was (5.55±1.50)%; when the incubation time was 60 min, the hemolysis rate was (16.55±5.57)%; when the incubation time was 90 min, the hemolysis rate was (55.73±5.17)%; when the incubation time was 120 min, the hemolysis rate was (96.4±4.49)%; when the incubation time was 150 min, the hemolysis rate was (97.5±4.30)%; when the incubation time was 180 min, the hemolysis rate was (100±0)%.

[0045] It can be seen from the above results that as the incubation time increases, the damaging effect of azobisisobutylamidine hydrochloride on red blood cells becomes more obvious and the hemolysis rate becomes higher.

[0046] 3. Protective effect of different concentrations of progesterone on hemolysis caused by 50mmol / lAPPH

[0047] After the samples of the APPH groups with different concentrations of progesterone were incubated at 37°C for 120 minutes, the hemolysis rates of each group were as follows:

[0048] When the progesterone is 0umol / l, the hemolysis rate is (61.74.58±4.71)%; when the progesterone is 1nmol / l, the hemolysis rate is (60.34±7.33)%; when the progesterone is 10nmol / l, the hemolysis rate is (42.83±4.29)%; when the progesterone is 30nmol / l, the hemolysis rate is (38.89±2.88)%; when the progesterone is 50nmol / l, the hemolysis rate is (18.75±1.67)%; when the progesterone is 100nmol / l, the hemolysis rate is (15.33±2.35)%.

[0049] From the above experimental results, it can be seen that when the progesterone protection concentration is ≥50nmol / l, the hemolysis rate of red blood cells caused by azobisisobutylamidine hydrochloride is significantly reduced, and the difference is statistically significant (P<0.05).

[0050] In summary, the progesterone concentration is greater than 10nmol / l, which has a significant protective effect on the hemolysis caused by APPH and is concentration-dependent. Based on this, the present invention establishes a progesterone non-genetic antioxidant stress model. The progesterone antioxidant stress model established by the method for establishing the progesterone non-genetic antioxidant stress model provided by the present invention can reflect the non-genetic antioxidant stress ability of progesterone in an objective and real way, solving the problem that the progesterone antioxidant stress model established by the method for establishing the progesterone antioxidant stress model in the prior art cannot objectively and truly reflect the non-genetic antioxidant stress ability of progesterone.

[0051] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0052] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0053] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for establishing a non-genetic anti-oxidative stress model of progesterone, characterized in that: The steps include: The ex vivo blood is centrifuged; after centrifugation, the red blood cell sediment in the lower layer is collected to obtain red blood cells; Take red blood cells, prepare a 5% volume percentage red blood cell suspension with PBS, add 1 nmol / L to 100 nmol / L progesterone and 30 mmol / L to 80 mmol / L azobisisobutylamidine hydrochloride to the red blood cell suspension, and finally obtain a red blood cell suspension containing progesterone and azobisisobutylamidine hydrochloride; heat the red blood cell suspension containing progesterone and azobisisobutylamidine hydrochloride, and centrifuge; take the supernatant after centrifugation, and measure the absorbance at 535 nm to 545 nm, which is recorded as A1'; Take red blood cells and prepare a 5% volume percentage red blood cell suspension containing only red blood cells with PBS; heat the 5% volume percentage red blood cell suspension containing only red blood cells and centrifuge it; take the supernatant after centrifugation and measure the absorbance at 535nm-545nm, which is recorded as A2'; According to the percentage of A1' and A2', the hemolysis rate of red blood cells was calculated to obtain the non-genetic antioxidant stress model of progesterone.

2. The method for establishing a progesterone non-genetic antioxidant stress model according to claim 1, characterized in that: The isolated blood is isolated carotid artery blood.

3. The method for establishing a progesterone non-genetic antioxidant stress model according to claim 1, characterized in that: The conditions for the in vitro blood centrifugation are: 2500 rpm to 3500 rpm, and centrifugation for 4 min to 6 min.

4. The method for establishing a progesterone non-genetic antioxidant stress model according to claim 1, characterized in that: The concentration of PBS is 0.005-0.015 M, and the pH is 7.3-7.

5.

5. The method for establishing a progesterone non-genetic antioxidant stress model according to claim 1, characterized in that: The concentration of the progesterone is 1.5 nmol / L to 99 nmol / L.

6. The method for establishing a progesterone non-genetic antioxidant stress model according to claim 5, characterized in that: The concentration of the progesterone is any one of 1 nmol / L, 10 nmol / L, 30 nmol / L, 50 nmol / L and 100 nmol / L.

7. The method for establishing a progesterone non-genetic antioxidant stress model according to claim 1, characterized in that: The heating time is 30 min to 180 min.

8. The method for establishing a progesterone non-genetic antioxidant stress model according to claim 1, characterized in that: A1' and A2' measure the absorbance at 540 nm.

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